U.S. patent application number 11/343557 was filed with the patent office on 2006-10-26 for novel compounds with high therapeutic index.
Invention is credited to V. Ravi Chandran.
Application Number | 20060241017 11/343557 |
Document ID | / |
Family ID | 37187695 |
Filed Date | 2006-10-26 |
United States Patent
Application |
20060241017 |
Kind Code |
A1 |
Chandran; V. Ravi |
October 26, 2006 |
Novel compounds with high therapeutic index
Abstract
The present invention is directed to novel therapeutic compounds
comprised of an amino acid bonded to a medicament or drug having a
hydroxy, amino, carboxy or acylating derivative thereon. These high
therapeutic index derivatives have the same utility as the drug
from which they are made, and they have enhanced pharmacological
and pharmaceutical properties. In fact, the novel drug derivatives
of the present invention enhance at least one therapeutic quality,
as defined herein. The present invention is also directed to
pharmaceutical compositions containing same.
Inventors: |
Chandran; V. Ravi; (Allen,
TX) |
Correspondence
Address: |
SCULLY SCOTT MURPHY & PRESSER, PC
400 GARDEN CITY PLAZA
SUITE 300
GARDEN CITY
NY
11530
US
|
Family ID: |
37187695 |
Appl. No.: |
11/343557 |
Filed: |
January 30, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US04/24901 |
Jul 29, 2004 |
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11343557 |
Jan 30, 2006 |
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60491331 |
Jul 29, 2003 |
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Current U.S.
Class: |
514/2.3 ;
514/16.3; 530/300 |
Current CPC
Class: |
A61P 29/00 20180101;
C07D 498/06 20130101; Y02A 50/30 20180101; C07C 229/08 20130101;
C07D 265/18 20130101; C07D 501/34 20130101; A61K 38/12 20130101;
C07D 239/10 20130101; C07D 295/185 20130101; C07D 493/04 20130101;
C07D 473/34 20130101; C07D 495/04 20130101; C07D 499/14 20130101;
A61K 47/542 20170801; C07D 311/32 20130101; C07D 401/12 20130101;
C07D 501/22 20130101; C07D 473/18 20130101; C07D 401/06 20130101;
C07D 413/06 20130101; C07D 498/14 20130101; C07F 9/572 20130101;
C07D 223/16 20130101; C07C 2602/22 20170501; Y02A 50/411 20180101;
C07D 261/20 20130101; C07D 311/24 20130101; C07D 333/58 20130101;
C07D 403/10 20130101; C07C 233/47 20130101; C07D 211/34 20130101;
C07D 493/10 20130101; C07D 209/10 20130101; C07D 209/14 20130101;
C07D 405/14 20130101; C07D 409/06 20130101; C07D 501/46 20130101;
C07F 9/65616 20130101; A61P 37/06 20180101; C07C 229/22 20130101;
C07C 229/26 20130101; C07D 207/33 20130101; C07C 229/36 20130101;
C07D 211/90 20130101; C07D 215/38 20130101; C07D 501/56 20130101;
C07D 217/26 20130101; C07D 275/04 20130101 |
Class at
Publication: |
514/002 ;
514/008; 530/300 |
International
Class: |
A61K 38/16 20060101
A61K038/16 |
Claims
1. A method for enhancing at least one of the therapeutic qualities
of a drug having a functionality group selected from the group
consisting of hydroxy, amino, carboxy or acylating derivatives of
said carboxy group, said improved therapeutic quality being
selected from the group consisting of: (a) improved taste or smell
(b) desired octanol/water partition coefficient (c) improved
stability (d) enhanced penetration of blood-brain barrier (e)
elimination of first pass effect in the liver (f) reduction of
intero-hepatic recirculation (g) painless injection with parental
formulation (h) improved bioavailability (i) improved changes in
the rate of absorption (j) reduced side effects (k) dose
proportionality (l) selective hydrolysis of the prodrug at site of
action (m) controlled release properties (n) targeted drug delivery
(o) reduction in toxicity (p) reduced dose (q) alteration of
metabolic pathway to deliver more drug at site of action (r)
increased solutility in aqueous solution and (s) enhanced efficacy,
the method comprising (a) reacting the drug with an amino acid
under conditions effective to form a covalent bond between the drug
and the amino acid and (b) administering the product of (a) to a
patient in need thereof, wherein said amino acid is a
.alpha.-L-amino acid.
2. The method according to claim 1 wherein the amino acid is a
naturally occurring amino acid.
3. The method according to claim 1 wherein the amino acid is Hyp,
Ser, Tyr, Lys, Leu, Ile, Gly, Asp, Glu, Met, Ala, Val, Pro, His,
Nor, Arg, Phe, Trp, Hsr, Car, Ort, Cav, Asn, Gln, Can, Tau, Djk,
GABA, Cys, Dcy and Sar.
4. The method according to claim 1 wherein the amino acid is Hyp,
Ser, Tyr, Lys, Leu, Ile, Gly, Asp, Glu, Met, Ala, Val, Pro, His,
Arg, Phe, Trp, Gln, Asn, or Cys.
5. The method according to any one of claim 1 wherein the the drug
is 5-HETE, Abacavir, Acarbose, Acebutolol, Acetaminophen, Adefovir,
Albuterol, Alfaprostol acid, Amlodipinel, Amoxicillin, Amphotericin
B, Amprenavir, Arachidonic Acid, Aspirin, Atenolol, Atorvastatin,
Atropine, Atovaquone, Baclofen, Benazeprilat, Beraprost, Betaxolol,
Bexarotene, Bicalutamide, Biotin, Biperiden, Bisoprolol,
Bitolterol, Brinzolamide, Bupivacaine, Buprenorphine, Bupropion,
Butorphanol, Candesartan, Capacitabine, Captopril, Carbidopa,
Carboprost, Carnitine, Carteolol, Carvedilol, Cefdinir, Cefditoren,
Ceftazimide, Cefpodoxime, Cefuroxime, Cerivastatin,
Chloramphenicol, Ciprofloxacin, Cisapride, Clofibrate, Clopidogrel
Acid, Cloprostenol, Clorazepic Acid, Cycloserine, Cyclosporine,
Cytarabine, Danazol, Dextroamphetamine, Diclofenac, Didanosine,
Digoxin, Divalproex, Docetaxel, Dorzolamide, Dyphylline,
Dysopyramide, Efavirenz, Enalaprilat, Ephedrine, Eplerenone,
Eprosartan, Esmolol, Estramustine, Ethambutol, Ethchlorvynol,
Ethosuximide, Ethotoin, Etidocaine, Etoposide, Ezetimibe,
Famciclovir, Fenofibrate, Fenoprofen, Fenprostalene acid,
Fexofenadine, Fibric acid derivatives, Finasteride, Flavoxate,
Fluprostenol, Fluoxetine, Flurbiprofen, Fluticasone, Fluvastatin,
Folic Acid, Fosinoprilat, Frovatriptan, Fulvestrant, Gabapentin,
Ganciclovir, Gemprost Acid, Glimepiride, Goserelin,
Hydroxychloroquine, Hydroxyzine, Hyoscyamine, Ibuprofen, Ibutilide,
Indapamide, Indinavir, Ipratropium, Irinotecan, Isosorbide,
Isradipine, Ketoprofen, Ketorolac, Labetalol, Lamivudine,
Lamivudine, Lansoprazole, Latanoprost Acid, Leukotrienes
(LTA.sub.4, LTB.sub.4, LTC.sub.4, LTD.sub.4 and LTE.sub.4)
Leuprolide, Levobunolol, Levodopa, Levorphanol, Limaprost,
.gamma.-Linolenic Acid, Liothyronine, Lisinopril, Lopinavir,
Lorazepam, Lovastatin, Medroxyprogesterone, Mefloquine, Megestrol,
Mephobarbital, Mepivacaine, Metaproterenol, Metformin,
Methamphetamine, Methohexital, Methotrexate, Methylphenidate,
Methylphenidate, Methylprednisolone, Metolazone, Metoprolol,
Mexiletine, Miglitol, Moexiprilat, Mometasone, Montelukast,
Mycophenolate Acid, Nadolol, Nalbuphine, Naproxen, Naratriptan,
Nateglinide, Nelfinavir, Niacin, Nicotinic Acid, Nicotinamide,
Nicardipine, Nimidipine, Nisoldipine, Norgestimate, Octreotide,
Ofloxacin, Olmesartan, Omeprazole, Ozagrel, Paclitaxel, Pantothenic
Acid, Paroxetine, Paroxetine, Pemoline, Penbutolol, Penicillamine,
Penciclovir, Pentazocine, Pentobarbital, Perindoprilat,
Phenylephrine, Phenylpropanolamine, Pindolol, Pioglitazone,
Pirbuterol, Pramipexole, Pravastatin, Propafenone, Propofol,
Propoxyphene, Propranolol, Prostacyclin, Prostaglandins (E.sub.1,
E.sub.2 and F.sub.2.alpha.), Prostanoic Acid, Pseudoephedrine,
Quinacrine, Quinaprilat, Quinethazone, Quinidine, Quinine,
Ramiprilat, Reboxetine, Repaglinide, Repaglinide, Ribavirin,
Ritonavir, Ropivacaine, Rosaprostol, Rosiglitazone, Rosuvastatin,
Salmeterol, Salsalate, Sertraline, Simavastatin, Sirolimus,
Sotalol, Sulfa Drugs, Sulfasalazine, Sumitriptan, Tacrolimus,
Tazorotene, Telmesartan, Tenofovir, Terbutaline, Thyroxine,
Tiagabine, Timolol, Tirofiban, Tocainide, Tramadol, Trandolaprilat,
Tranylcypromine, Treprostinil, Triamcinolone, Trimoprostil,
Troglitazone, Unoprostone, Valproic Acid, Valsartan, Venlafaxine,
Vidarabine, Warfarin, Zalcitabine, Zidovudine, Zileuton and
Zolmitriptan or a pharmaceutically acceptable salt of any of said
drug.
6. The method according to claim 1 wherein AA is Tyrosine,
hydroxyproline or Serine.
7. The product from a reaction between a drug having a
functionality selected from the group consisting of hydroxy, amino,
carboxy or acylating derivative of said carboxy group and a
L-.alpha.-amino acid under conditions sufficient to form a covalent
bond from the reaction of said functional group and said amino
acid, or the pharmaceutically acceptable salt thereof, wherein the
drug is selected from the group consisting of 5-HETE, Abacavir,
Acarbose, Acebutolol, Acetaminophen, Adefovir, Albuterol,
Alfaprostol acid, Amlodipinel, Amoxicillin, Amphotericin B,
Amprenavir, Arachidonic Acid, Aspirin, Atenolol, Atorvastatin,
Atropine, Atovaquone, Baclofen, Benazeprilat, Beraprost, Betaxolol,
Bexarotene, Bicalutamide, Biotin, Biperiden, Bisoprolol,
Bitolterol, Brinzolamide, Bupivacaine, Buprenorphine, Bupropion,
Butorphanol, Candesartan, Capacitabine, Captopril, Carbidopa,
Carboprost, Carnitine, Carteolol, Carvedilol, Cefdinir, Cefditoren,
Ceftazimide, Cefpodoxime, Cefuroxime, Cerivastatin,
Chloramphenicol, Ciprofloxacin, Cisapride, Clofibrate, Clopidogrel
Acid, Cloprostenol, Clorazepic Acid, Cycloserine, Cyclosporine,
Cytarabine, Danazol, Dextroamphetamine, Diclofenac, Didanosine,
Digoxin, Divalproex, Docetaxel, Dorzolamide, Dyphylline,
Dysopyramide, Efavirenz, Enalaprilat, Ephedrine, Eplerenone,
Eprosartan, Esmolol, Estramustine, Ethambutol, Ethchlorvynol,
Ethosuximide, Ethotoin, Etidocaine, Etoposide, Ezetimibe,
Famciclovir, Fenofibrate, Fenoprofen, Fenprostalene acid,
Fexofenadine, Fibric acid derivatives, Finasteride, Flavoxate,
Fluprostenol, Fluoxetine, Flurbiprofen, Fluticasone, Fluvastatin,
Folic Acid, Fosinoprilat, Frovatriptan, Fulvestrant, Gabapentin,
Ganciclovir, Gemprost Acid, Glimepiride, Goserelin,
Hydroxychloroquine, Hydroxyzine, Hyoscyamine, Ibuprofen, Ibutilide,
Indapamide, Indinavir, Ipratropium, Irinotecan, Isosorbide,
Isradipine, Ketoprofen, Ketorolac, Labetalol, Lamivudine,
Lamivudine, Lansoprazole, Latanoprost Acid, Leukotrienes
(LTA.sub.4, LTB.sub.4, LTC.sub.4, LTD.sub.4 and LTE.sub.4)
Leuprolide, Levobunolol, Levodopa, Levorphanol, Limaprost,
.gamma.-Linolenic Acid, Liothyronine, Lisinopril, Lopinavir,
Lorazepam, Lovastatin, Medroxyprogesterone, Mefloquine, Megestrol,
Mephobarbital, Mepivacaine, Metaproterenol, Metformin,
Methamphetamine, Methohexital, Methotrexate, Methylphenidate,
Methylphenidate, Methylprednisolone, Metolazone, Metoprolol,
Mexiletine, Miglitol, Moexiprilat, Mometasone, Montelukast,
Mycophenolate Acid, Nadolol, Nalbuphine, Naproxen, Naratriptan,
Nateglinide, Nelfinavir, Niacin, Nicotinic Acid, Nicotinamide,
Nicardipine, Nimidipine, Nisoldipine, Norgestimate, Octreotide,
Ofloxacin, Olmesartan, Omeprazole, Ozagrel, Paclitaxel, Pantothenic
Acid, Paroxetine, Paroxetine, Pemoline, Penbutolol, Penicillamine,
Penciclovir, Pentazocine, Pentobarbital, Perindoprilat,
Phenylephrine, Phenylpropanolamine, Pindolol, Pioglitazone,
Pirbuterol, Pramipexole, Pravastatin, Propafenone, Propofol,
Propoxyphene, Propranolol, Prostacyclin, Prostaglandins (E.sub.1,
E.sub.2 and F.sub.2.alpha.), Prostanoic Acid, Pseudoephedrine,
Quinacrine, Quinaprilat, Quinethazone, Quinidine, Quinine,
Ramiprilat, Reboxetine, Repaglinide, Repaglinide, Ribavirin,
Ritonavir, Ropivacaine, Rosaprostol, Rosiglitazone, Rosuvastatin,
Salmeterol, Salsalate, Sertraline, Simavastatin, Sirolimus,
Sotalol, Sulfa Drugs, Sulfasalazine, Sumitriptan, Tacrolimus,
Tazorotene, Telmesartan, Tenofovir, Terbutaline, Thyroxine,
Tiagabine, Timolol, Tirofiban, Tocainide, Tramadol, Trandolaprilat,
Tranylcypromine, Treprostinil, Triamcinolone, Trimoprostil,
Troglitazone, Unoprostone, Valproic Acid, Valsartan, Venlafaxine,
Vidarabine, Warfarin, Zalcitabine, Zidovudine, Zileuton and
Zolmitriptan or a pharmaceutically acceptable salt of any of said
drug and wherein the amino acid is Hyp, Ser, Tyr, Lys, Leu, Ile,
Gly, Asp, Glu, Met, Ala, Val, Pro, His, Nor, Arg, Phe, Trp, Hsr,
Car, Ort, Cav, Asn, Gln, Can, Tau, Djk, GABA, Cys, Sar or Dcy.
8. The product according to claim 7 wherein the product is:
##STR144## ##STR145## ##STR146## ##STR147## ##STR148## ##STR149##
##STR150## R.sub.8N.dbd.N-AA.sub.5-COOH, SULFO DRUG AZO DERIVATIVE,
R.sub.8NH CO-AA.sub.5NH.sub.2 SULFA DRUG AMIDE DERIVATIVE or
##STR151## or a pharmaceutically acceptable salt thereof, wherein
AA is an amino acid without the hydroxyl group on the carboxy
group, R is NHAA or OAA.sub.1, CYCLO represents the residues at
positions 2-11 of a cyclosporin molecule; x-y is CH.dbd.CH or
CH.sub.2.dbd.CH.sub.2; R.sup.2 is OAA or OGlyAA; AA.sub.1 is an
amino acid without the hydroxy group on the carboxy group; AA.sub.6
is an amino acid residue having a hydroxy group on the side less
said hydroxyl group, and wherein AA.sub.6 is attached via an ester
bond through its side chain hydroxy group; R.sub.2 is ##STR152##
R.sub.4 is NHAA or OAA.sub.1; R.sub.5 is AA.sub.2; AA.sub.2 is an
amino acid residue less the hydroxy group on the carboxy group;
AA.sub.3 is an amino acid less the hydrogen atom or the amino
group; R.sub.3 is ethyl; R.sub.1 is AA without the carboxy and
amino groups; R.sub.6 is COAA.sub.1; R.sub.7 is O-AA.sub.1;
AA.sub.4 is an amino acid residue wherein it is attached via an
ester bond through the acyl group of the carboxy group; AA.sub.5 is
an amino acid without the amino and carboxy group; R.sub.8 is a
sulfanilamide moiety of the general class of sulfa drugs; and
R.sub.9 is NHAA or OAA.sub.6, and AA, AA.sub.1, AA.sub.2, AA.sub.3,
AA.sub.4, and AA.sub.5 are independently L-amino acids and are Thr,
Hyp, Ser, Tyr, Lys, Leu, Ile, Gly, Asp, Glu, Met, Val, Pro, His,
Nor, Arg, Phe, Trp, Hsr, Car, Ort, Cys, Cav, Asn, Gln, Can, Tau,
Djk, GABA, Dcy or Sar, and AA.sub.6 is an L-amino acid selected
from Hyp, Ser or Tyr.
9. The product according to claim 8 wherein AA.sub.1, AA.sub.2,
AA.sub.3, AA.sub.4, and AA.sub.5 are independently Ser, Tyr, Lys,
Leu, Ile, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Arg, Phe, Trp,
Gln, Asn, Cys or Ser.
10. The method according to claim 9 wherein AA.sub.1, AA.sub.2,
AA.sub.3, AA.sub.4, AA.sub.5 and AA.sub.6 are independently
Tyrosine, hydroxyproline or serine.
11. The compound according to claim 10 wherein AA.sub.1, AA.sub.2,
AA.sub.3, AA.sub.4, AA.sub.5 and AA.sub.6 are hydroxyproline.
12. The compound according to claim 8 wherein the compound has the
formula ##STR153##
13. A pharmaceutical composition comprising a therapeutically
effective amount of a compound according to claim 7 and a
pharmaceutically acceptable salt thereof.
14. A method of enhancing the bioavailability of a drug having a
functionally selected from the group consisting of hydroxy, amino,
carboxy or acylating derivative of said carboxy group, said drug
being selected from the group consisting of 5-HETE, Abacavir,
Acarbose, Acebutolol, Acetaminophen, Adefovir, Albuterol,
Alfaprostol acid, Amlodipinel, Amoxicillin, Amphotericin B,
Amprenavir, Arachidonic Acid, Aspirin, Atenolol, Atorvastatin,
Atropine, Atovaquone, Baclofen, Benazeprilat, Beraprost, Betaxolol,
Bexarotene, Bicalutamide, Biotin, Biperiden, Bisoprolol,
Bitolterol, Brinzolamide, Bupivacaine, Buprenorphine, Bupropion,
Butorphanol, Candesartan, Capacitabine, Captopril, Carbidopa,
Carboprost, Carnitine, Carteolol, Carvedilol, Cefdinir, Cefditoren,
Ceftazimide, Cefpodoxime, Cefuroxime, Cerivastatin,
Chloramphenicol, Ciprofloxacin, Cisapride, Clofibrate, Clopidogrel
Acid, Cloprostenol, Clorazepic Acid, Cycloserine, Cyclosporine,
Cytarabine, Danazol, Dextroamphetamine, Diclofenac, Didanosine,
Digoxin, Divalproex, Docetaxel, Dorzolamide, Dyphylline,
Dysopyramide, Efavirenz, Enalaprilat, Ephedrine, Eplerenone,
Eprosartan, Esmolol, Estramustine, Ethambutol, Ethchlorvynol,
Ethosuximide, Ethotoin, Etidocaine, Etoposide, Ezetimibe,
Famciclovir, Fenofibrate, Fenoprofen, Fenprostalene acid,
Fexofenadine, Fibric acid derivatives, Finasteride, Flavoxate,
Fluprostenol, Fluoxetine, Flurbiprofen, Fluticasone, Fluvastatin,
Folic Acid, Fosinoprilat, Frovatriptan, Fulvestrant, Gabapentin,
Ganciclovir, Gemprost Acid, Glimepiride, Goserelin,
Hydroxychloroquine, Hydroxyzine, Hyoscyamine, Ibuprofen, Ibutilide,
Indapamide, Indinavir, Ipratropium, Irinotecan, Isosorbide,
Isradipine, Ketoprofen, Ketorolac, Labetalol, Lamivudine,
Lamivudine, Lansoprazole, Latanoprost Acid, Leukotrienes
(LTA.sub.4, LTB.sub.4, LTC.sub.4, LTD.sub.4 and LTE.sub.4)
Leuprolide, Levobunolol, Levodopa, Levorphanol, Limaprost,
.gamma.-Linolenic Acid, Liothyronine, Lisinopril, Lopinavir,
Lorazepam, Lovastatin, Medroxyprogesterone, Mefloquine, Megestrol,
Mephobarbital, Mepivacaine, Metaproterenol, Metformin,
Methamphetamine, Methohexital, Methotrexate, Methylphenidate,
Methylphenidate, Methylprednisolone, Metolazone, Metoprolol,
Mexiletine, Miglitol, Moexiprilat, Mometasone, Montelukast,
Mycophenolate Acid, Nadolol, Nalbuphine, Naproxen, Naratriptan,
Nateglinide, Nelfinavir, Niacin, Nicotinic Acid, Nicotinamide,
Nicardipine, Nimidipine, Nisoldipine, Norgestimate, Octreotide,
Ofloxacin, Olmesartan, Omeprazole, Ozagrel, Paclitaxel, Pantothenic
Acid, Paroxetine, Paroxetine, Pemoline, Penbutolol, Penicillamine,
Penciclovir, Pentazocine, Pentobarbital, Perindoprilat,
Phenylephrine, Phenylpropanolamine, Pindolol, Pioglitazone,
Pirbuterol, Pramipexole, Pravastatin, Propafenone, Propofol,
Propoxyphene, Propranolol, Prostacyclin, Prostaglandins (E.sub.1,
E.sub.2 and F.sub.2.alpha.), Prostanoic Acid, Pseudoephedrine,
Quinacrine, Quinaprilat, Quinethazone, Quinidine, Quinine,
Ramiprilat, Reboxetine, Repaglinide, Repaglinide, Ribavirin,
Ritonavir, Ropivacaine, Rosaprostol, Rosiglitazone, Rosuvastatin,
Salmeterol, Salsalate, Sertraline, Simavastatin, Sirolimus,
Sotalol, Sulfa Drugs, Sulfasalazine, Sumitriptan, Tacrolimus,
Tazorotene, Telmesartan, Tenofovir, Terbutaline, Thyroxine,
Tiagabine, Timolol, Tirofiban, Tocainide, Tramadol, Trandolaprilat,
Tranylcypromine, Treprostinil, Triamcinolone, Trimoprostil,
Troglitazone, Unoprostone, Valproic Acid, Valsartan, Venlafaxine,
Vidarabine, Warfarin, Zalcitabine, Zidovudine, Zileuton and
Zolmitriptan, said method comprising reacting the drug with an
amino acid under conditions effective to form a covalent bond
between the drug and the amino acid and administering the product
thereof to a patient in need thereof, wherein said amino acid is a
L-amino acid selected from the groups consisting of Hyp, Ser, Tyr,
Lys, Leu, Ile, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Nor, Ara,
Phe, Trp, Hsr, Car, Ort, Cav, Asn, Gln, Can, Tau, Djk, GABA, Cys,
Sar or Dcy.
15. A method of enhancing the solubility in an aqueous solution of
a drug having a functionality selected from the group consisting of
hydroxy, amino, carboxy or acylating derivative of said carboxy
group, said drug being selected from the group consisting of
5-HETE, Abacavir, Acarbose, Acebutolol, Acetaminophen, Adefovir,
Albuterol, Alfaprostol acid, Amlodipinel, Amoxicillin, Amphotericin
B, Amprenavir, Arachidonic Acid, Aspirin, Atenolol, Atorvastatin,
Atropine, Atovaquone, Baclofen, Benazeprilat, Beraprost, Betaxolol,
Bexarotene, Bicalutamide, Biotin, Biperiden, Bisoprolol,
Bitolterol, Brinzolamide, Bupivacaine, Buprenorphine, Bupropion,
Butorphanol, Candesartan, Capacitabine, Captopril, Carbidopa,
Carboprost, Carnitine, Carteolol, Carvedilol, Cefdinir, Cefditoren,
Ceftazimide, Cefpodoxime, Cefuroxime, Cerivastatin,
Chloramphenicol, Ciprofloxacin, Cisapride, Clofibrate, Clopidogrel
Acid, Cloprostenol, Clorazepic Acid, Cycloserine, Cyclosporine,
Cytarabine, Danazol, Dextroamphetamine, Diclofenac, Didanosine,
Digoxin, Divalproex, Docetaxel, Dorzolamide, Dyphylline,
Dysopyramide, Efavirenz, Enalaprilat, Ephedrine, Eplerenone,
Eprosartan, Esmolol, Estramustine, Ethambutol, Ethchlorvynol,
Ethosuximide, Ethotoin, Etidocaine, Etoposide, Ezetimibe,
Famciclovir, Fenofibrate, Fenoprofen, Fenprostalene acid,
Fexofenadine, Fibric acid derivatives, Finasteride, Flavoxate,
Fluprostenol, Fluoxetine, Flurbiprofen, Fluticasone, Fluvastatin,
Folic Acid, Fosinoprilat, Frovatriptan, Fulvestrant, Gabapentin,
Ganciclovir, Gemprost Acid, Glimepiride, Goserelin,
Hydroxychloroquine, Hydroxyzine, Hyoscyamine, Ibuprofen, Ibutilide,
Indapamide, Indinavir, Ipratropium, Irinotecan, Isosorbide,
Isradipine, Ketoprofen, Ketorolac, Labetalol, Lamivudine,
Lamivudine, Lansoprazole, Latanoprost Acid, Leukotrienes
(LTA.sub.4, LTB.sub.4, LTC.sub.4, LTD.sub.4 and LTE.sub.4)
Leuprolide, Levobunolol, Levodopa, Levorphanol, Limaprost,
.gamma.-Linolenic Acid, Liothyronine, Lisinopril, Lopinavir,
Lorazepam, Lovastatin, Medroxyprogesterone, Mefloquine, Megestrol,
Mephobarbital, Mepivacaine, Metaproterenol, Metformin,
Methamphetamine, Methohexital, Methotrexate, Methylphenidate,
Methylphenidate, Methylprednisolone, Metolazone, Metoprolol,
Mexiletine, Miglitol, Moexiprilat, Mometasone, Montelukast,
Mycophenolate Acid, Nadolol, Nalbuphine, Naproxen, Naratriptan,
Nateglinide, Nelfinavir, Niacin, Nicotinic Acid, Nicotinamide,
Nicardipine, Nimidipine, Nisoldipine, Norgestimate, Octreotide,
Ofloxacin, Olmesartan, Omeprazole, Ozagrel, Paclitaxel, Pantothenic
Acid, Paroxetine, Paroxetine, Pemoline, Penbutolol, Penicillamine,
Penciclovir, Pentazocine, Pentobarbital, Perindoprilat,
Phenylephrine, Phenylpropanolamine, Pindolol, Pioglitazone,
Pirbuterol, Pramipexole, Pravastatin, Propafenone, Propofol,
Propoxyphene, Propranolol, Prostacyclin, Prostaglandins (E.sub.1,
E.sub.2 and F.sub.2.alpha.), Prostanoic Acid, Pseudoephedrine,
Quinacrine, Quinaprilat, Quinethazone, Quinidine, Quinine,
Ramiprilat, Reboxetine, Repaglinide, Repaglinide, Ribavirin,
Ritonavir, Ropivacaine, Rosaprostol, Rosiglitazone, Rosuvastatin,
Salmeterol, Salsalate, Sertraline, Simavastatin, Sirolimus,
Sotalol, Sulfa Drugs, Sulfasalazine, Sumitriptan, Tacrolimus,
Tazorotene, Telmesartan, Tenofovir, Terbutaline, Thyroxine,
Tiagabine, Timolol, Tirofiban, Tocainide, Tramadol, Trandolaprilat,
Tranylcypromine, Treprostinil, Triamcinolone, Trimoprostil,
Troglitazone, Unoprostone, Valproic Acid, Valsartan, Venlafaxine,
Vidarabine, Warfarin, Zalcitabine, Zidovudine, Zileuton and
Zolmitriptan, said method comprising reacting the covalent bond
with an amino acid under conditions effective to form a drug
between the drug and the amino acid and administering the product
thereof to a patient in need thereof, wherein said amino acid is a
L-amino acid selected from the group consisting of Hyp, Ser, Tyr,
Lys, Leu, Ile, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Nor, Ara,
Phe, Trp, Hsr, Car, Ort, Cav, Asn, Gln, Can, Tau, Djk, GABA, Cys,
Sar or Dcy.
16. The method according to claim 14 wherein the amino acid is Hyp,
Ser, Tyr, Lys, Leu, Ile, Gly, Asp, Glu, Met, Ala, Val, Pro, His,
Arg, Phe, Trp, Gln, Asn, or Cys.
17. The method according to claim 15 wherein the amino acid is Hyp,
Ser, Tyr, Lys, Leu, Ile, Gly, Asp, Glu, Met, Ala, Val, Pro, His,
Arg, Phe, Trp, Gln, Asn, or Cys.
18. The method according to claim 14 wherein the amino acid is
hydroxyproline, tyrosine or serine.
19. The method according to claim 15 wherein the amino acid is
hydroxyproline, tyrosine or serine.
20. A method of treating infectious diseases in mammals casued by
pathogenic microorganisms, said method comprising administering to
said mammal in need of treatment a therapeutically effective amount
of a compound prepared by reacting the carboxylic acid
functionality of the cefdinir molecule with an amino acid or
acylating derivative under ester or amide forming conditions and
isolating the product thereof.
21. A method of enhancing the safety profile with longer anesthetic
effect of propofol in an aqueous solution comprising reacting the
hydroxy functionality of the propofol molecule with an amino acid
or acylating derivative thereof under ester forming conditions and
isolating the product thereof.
22. A method of enhancing the solubility of a quinolone antibiotic
having a carboxy group or acylating group in an aqueous solution
comprising reacting the carboxylic acid functionality of the
quinolone antibiotic with an amino acid or acylating derivative
thereof under either ester or amide forming conditions and
isolating the product thereof.
23. A method of enhancing the bioavailability of a quinolone
antibiotic having a carboxyl group or acylating group when
administered to a patient which comprises reacting the carboxylic
acid functionality of the quinolone antibiotic with an amino acid
or acylating derivative under ester or amide forming conditions,
isolating the product thereof and administering said product to the
patient.
24. A method of enhancing the solubility of ACE inhibitors having a
carboxy or acylating group in an aqueous solution comprising
reacting the carboxylic acid functionality of the ACE inhibitor
molecule with an amino acid or acylating derivative thereof under
either ester or amide forming conditions and isolating the product
thereof.
25. A method of enhancing the bioavailability of ACE inhibitors
having a carboxy or acylating group when administered to a patient
which comprises reacting the carboxylic acid functionality of the
ACE inhibitors molecule with an amino acid or acylating derivative
under ester or amide forming conditions, isolating the product
thereof and administering said product to the patient.
26. A method of treating a patient infected with a microorganism to
which a drug selected from the group consisting of amoxicillin,
cefuroxime, ceftazidime and cexpodoxine is toxic, said method
comprising administering to said patient a therapeutically
effective amount of the compound prepared by reacting the drug with
an amino acid or acylating derivative thereof under either ester or
amide forming conditions and isolating the products thereof.
27. A method of reducing the gastric irritation of ibuprofen
comprising reacting the carboxylic acid functionality of the
ibuprofen molecule with an amino acid or acylating derivative under
either ester or amide forming conditions and administering the
product to a patient in need thereof, said product resulting less
gastric irritability then ibuprofen.
28. A method of enhancing the solubility of a proton pump inhibitor
in an aqueous solution comprising reacting the amine functionality
of the proton pump inhibitor with the carboxylic acid moiety of the
amino acid or acylating derivative thereof under amide forming
conditions and isolating the product thereof.
29. A method of enhancing the bioavailability of a proton pump
inhibitor when administered to a patient which comprises reacting
the amine functionality of the proton pump inhibitor with the
carboxylic acid moiety of an amino acid or acylating derivative
thereof under amide forming conditions, isolating the product
thereof and administering said product to the patient.
30. A method of enhancing the solubility of Selective 5-HT receptor
agonists in an aqueous solution comprising reacting the amine
functionality of the Selective 5-HT receptor agonists molecule with
the carboxylic acid moiety of an amino acid or acylating derivative
thereof under amide forming conditions and isolating the product
thereof.
31. A method of enhancing the bioavailability of Selective 5-HT
receptor agonists when administered to a patient which comprises
reacting the amine functionality of the Selective 5-HT receptor
agonists with the carboxylic acid moiety of an amino acid under
amide forming conditions, isolating the product thereof and
administering said product to the patient.
32. A method of reducing the gastric irritability in the stomach
resulting from aspirin administration to a mammal which method
comprises reacting the carboxylic acid functionality of the aspirin
molecule with an amino acid or acylating derivative thereof under
either amide or ester forming conditions, and administering the
formed product to said mammal, wherein the gastric irritability is
reduced relative to aspirin.
33. A method of enhancing the solubility of immunosuppresants in an
aqueous solution comprising reacting the hydroxy functionality of
the immunosuppresants with an amino acid or acylating derivative
thereof under ester forming conditions and isolating the product
thereof.
34. A method of enhancing the bioavailability of immunosuppresants
when administered to a patient which comprises reacting the hydroxy
functionality of the immunosuppresants with an amino acid or
acylating derivative under ester forming conditions, isolating the
product thereof and administering said product to the patient.
35. A method of enhancing the solubility of a sulfa drug in an
aqueous solution comprising reacting the amine of the sulfa drug
with an amino acid or acylating derivative thereof under either azo
or amide forming conditions and isolating product thereof.
36. A method of enhancing the bioavailabilty of a sulfa drug when
administered to a patient which comprises reacting the amine
functionality of the sulfa drug with an amino acid or acylating
derivative under azo or amide forming conditions, isolating the
product thereof and administering said product to the patient.
37. A method of providing a clear aqueous intravenous formulation
comprising a Sulfa drug when administered to a patient which
comprises reacting the amine of the Sulfa drug with an amino acid
or acylating derivative under azo or amide forming conditions, and
isolating the product thereof said product being prepared in an
aqueous non-toxic pharmaceutically acceptable solvent or being
added, after the formation thereof to an aqueous non-toxic
pharmaceutically acceptable solvent.
38. A method of enhancing the solubility in an aqueous solution of
a nucleoside analog having an OH functionality comprising reacting
the hydroxyl functionality of the nucleoside analog with an amino
acid or acylating derivative thereof under ester forming conditions
and isolating the product thereof.
39. A method for enhancing the bioavailability of a nucleoside
analog having an OH group thereon which comprise reacting the
hydroxyl functionality of the nucleoside analog with an amino acid
or acylating derivative thereof under under ester forming
conditions and isolating the product thereof and administering the
product to a patient.
Description
RELATED APPLICATION
[0001] This is a continuation-in-part of PCT Application No.
PCT/US04/24901 which claims benefit of provisional application No.
60/491,331 filed on Jul. 29, 2003.
SCOPE OF THE INVENTION
[0002] The invention relates to amino acid derivatives of
pharmaceutical compounds, methods of treating particular ailments,
which are ameliorated by the administration of these drugs and
pharmaceutical compositions containing these drugs.
The current invention involves improving many physicochemical,
biopharmaceutical, and clinical efficacy of various drugs using
amino acids as covalently bonded carriers for these drugs.
BACKGROUND OF THE INVENTION
[0003] The development of chemical compounds for the treatment of
disorders, maladies and diseases has become increasingly difficult
and costly. The probability of success for such development is
often discouragingly low. Further, the time for development can
approach or exceed ten years, leaving large numbers of patients
without remedy for an extended period of time.
[0004] Even in cases in which effective pharmaceutical compounds
have been developed, there are often disadvantages associated with
their administration. These disadvantages can include aesthetic,
and pharmacokinetic barriers affecting the effectiveness of some
existing pharmaceutical compounds, For example, unpleasant taste or
smell of a pharmaceutical compound or composition can be a
significant barrier to patient compliance with respect to the
administration regimen. The undesirable solubility characteristics
of a pharmaceutical compound can lead to difficulty in formulation
of a homogeneous composition. Other disadvantages associated with
known pharmaceutical compounds include: poor absorption of orally
administered formulations; poor bioavailability of the
pharmaceutical compounds in oral and other non-intravenous
formulations; lack of dose proportionality; low stability of
pharmaceutical compounds in vitro and in vivo; poor penetration of
the blood/brain barrier; excessive first-pass metabolism of
pharmaceutical compounds as they pass through the liver; excessive
enterohepatic recirculation; low absorption rates from various
sites, including skin, rectal, oral and buccal administration;
ineffective compound release at the site of action; excessive
irritation, for example, gastro-intestinal irritability and/or
ulceration; painful injection of parenterally administered
pharmaceutical compounds and compositions; excessively high dosages
required for some pharmaceutical compounds and compositions, and
other undesirable characteristics. Some pharmaceutical compounds
are processed by the body to produce toxic by-products with harmful
effects.
[0005] The art is continually seeking new chemical compounds for
the treatment of a wide variety of disorders, with improved
properties to overcome the disadvantages of known pharmaceutical
compounds mentioned above.
[0006] The present invention has overcome many problems associated
with currently marketed drugs by making amino acid derivatives. The
concept of such derivative chemistry is well known, and there are a
number of examples of such derivatives enumerated in the literature
and there and even a few such derivatives are available in the
market, including such diverse groups as statin drugs, ACE
inhibitors, antiviral drugs such as Acyclovir and the like.
SUMMARY OF THE PRESENT INVENTION
[0007] The present invention, however, uses amino acids as the
moiety to make such derivatives. The inventor has found that the
novel drug derivatives of the present invention have a number of
advantages. For example, when the amino acid derivatives of the
present invention are administered by a number of routes such as
oral, IV, rectal or other such methods, the inventor has found that
generally these derivatives are not broken down in vivo; rather the
amino acid derivatives themselves possess the therapeutic
activity.
[0008] In many cases, the inventor found that the current amino
acid derivatives do not dissociate into the drug from which the
amino acid derivative is derived, since there appears to be no
active drug in the systemic circulation. Instead, what was found in
the systemic circulation was the amino acid derivative is active
and less toxic than the corresponding drug and possesses high
therapeutic index. A significant advantage of the amino acid
derivative of the present invention is that it is non-toxic, and
hence either assimilated into the body or safely excreted. This is
quite unlike the characteristics exhibited by a number of other
drug derivatives available in the market, where the promoiety
itself is toxic, as is the case with statin drugs, Enalapril,
Benazapril and the like group of ACE inhibitors, and a number of
antibiotics such as pivoxil, Axetil, Cilexetil and the like groups,
which are highly toxic, thereby reducing the therapeutic index of
the active drug.
[0009] On the other hand, the amino acid derivatives of the present
invention also impart a number of advantages as shown herein
below.
[0010] The present invention is directed to pharmaceutically active
drugs, having an amino acid covalently bonded thereto to form said
amino acid derivative, which is administered in this form to the
subject, such as a mammal.
[0011] The amino acid is an ideal model to be used as a derivative,
because it is capable of forming various types of linkages between
itself and the drug. By definition, an amino acid has at least two
functionalities thereon, an amino group (NH.sub.2) and a carboxy
group (COOH). For example, the .alpha.-amino acids have the well
known structure ##STR1## wherever R.sub.0 is the side group or
chain of the amino acid. The ##STR2## as defined herein, is the
main chain of the amino acid. Thus, for example, in addition to the
amino group and the carboxyl group on the main chain, the side
chain may have other functional groups thereon such as SH or OH or
NH.sub.2 and the like. The functional groups on the amino acid
moiety permit the covalent linkage to occur between the amino acid
and the drug.
[0012] As defined herein, the drug or medicament useful in the
present invention contains functional groups thereon that permit
the drug to react with and form a covalent bond with the amino
acid. Examples of functional groups present on the drugs include
NH.sub.2, OH, COOH or acylating derivatives (acid derivatives)
thereof, such as esters, amides, anhydrides and the like.
[0013] The mode of attachment between the pharmaceutical compound
and the amino acid can be via:
[0014] 1) An ester bond (--CO--O--) arising from condensation of a
carboxylic acid and an alcohol or phenolic hydroxyl group, or
through transesterification, for example: [0015] a) Where the
pharmaceutical compound has an aliphatic or aromatic hydroxyl
group, an ester bond can be formed with the backbone carboxylic
acid group of the amino group under esterification conditions; or
[0016] b) Where the pharmaceutical compound has an aliphatic or
aromatic hydroxyl group and the amino acid has a side chain
carboxylic acid group, an ester bond can be formed therebetween
under esterification conditions; or [0017] c) Where the
pharmaceutical compound has a carboxylic acid group and the amino
acid has a side chain aliphatic or aromatic hydroxyl group, an
ester bond can be formed therebetween under esterification
condition; or [0018] d) Where the pharmaceutical compound has an
ester group with substituted or unsubstituted acyloxy (e.g.,
carbonylalkoxy or arylalkoxycarbonyl, or aryloxycarbonyl)
substituent (compound-O--CO-substituent) and the amino acid has a
backbone carboxylic acid group, an ester bond can be formed
therebetween through transesterification; or [0019] e) Where the
pharmaceutical compound has an ester group with a substituted or
unsubstituted acyloxy (e.g., alkoxycarbonyl or arylalkoxycarbonyl,
or aryloxy carbonyl) substituent (compound-O--CO-substituent) and
the amino acid has a side chain carboxylic acid group, an ester
bond can be formed therebetween through transesterification; or
[0020] f) Where the pharmaceutical compound has an ester group with
a substituted or unsubstituted alkoxycarbonyl or arylalkoxycarbonyl
or aryloxy carbonyl substituent (compound-CO--O-Substituent) and
the amino acid has a side chain aliphatic or aromatic hydroxyl
group, an ester bond can form therebetween though
transesterification; or [0021] g) The alcohol and carboxylic acid
moieties may be on the same molecule such they can form an internal
ester. For example, certain compounds like Gabapentin can form an
internal ester under ester forming conditions, and is also included
within the scope of the present invention.
[0022] 2) An amide bond (--CO--NH--) arising from condensation of a
carboxylic acid and an amine, for example: [0023] a) Where the
pharmaceutical compound has an amino group and the amino acid has a
backbone carboxylic acid group, an amide can be formed under amide
forming conditions; or [0024] b) Where the pharmaceutical compound
has an amino group and the amino acid has a side chain carboxylic
acid group, an amide bond can form therebetween under amide forming
conditions; or [0025] c) Where the pharmaceutical compound has a
carboxylic acid group and the amino acid has a backbone amino
group, an amide bond can form therebetween under amide forming
conditions; or [0026] d) Where the pharmaceutical compound has a
carboxylic acid group and the amino acid has a side chain amino
group, an amide bond can be formed therebetween under amide forming
conditions.
[0027] Thus, the present invention is directed to the amino acid
derivatives thus formed. As shown hereinbelow, the amino acid
derivatives described herein have advantages not realized relative
to the drug without the amino acid attached thereto. For example,
it can improve bioavailability, efficacy, be less toxic, exhibit
greater solubility in water and/or improve the ability of the drug
to pass into the cell membrane or through blood brain barrier,
exhibit less side effects, such as gastrointestinal irritability,
enhanced therapeutic index and the like.
[0028] Thus, the present invention is directed to a method of
improving the therapeutic quality of a drug wherein the improvement
in the therapeutic quality is selected from the group consisting of
improved efficacy, enhanced therapeutic index, increased solubility
in the mammal's internal fluid, improved passage through the cell
membrane, improved passage through the blood brain barrier,
decreased side effects, such as significantly decreased irritation
and/or ulcerations, less toxicity, enhanced absorption ratio and
the like relative to the corresponding drug administered to the
patient in the derivatized form, said method comprising reacting
the drug with an amino acid to form a covalent bond therebetween
and administering the product thereof (hereinafter "derivative") to
a patient. The amino acid derivatives of the present invention have
at least one improved quality. In fact, they exhibit preferably at
least one of the improved qualities cited hereinabove, and more
preferably, at least two of the qualities described herein. Other
advantages of the derivative include the wide availability of the
amino acids and the ease in which the reactions take place. The
reaction to form the amide and esters are generally efficient and
yields are very high, presumably above about 60% and more
preferably above about 75% and most preferably above about 90%.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The file of this patent application contains at least one
drawings executed in color. Copies of this patent application with
color drawing(s) will be provided by the United States Patent and
Trademark Office upon request of the necessary fee.
[0030] FIG. 1 graphically compares the efficacy of L-serine ester
of (.+-.) Ibuprofen (F1), L-threonine ester (.+-.) Ibuprofen (F2)
and L-hydroxyproline ester of (.+-.) Ibuprofen (F3), (.+-.)
Ibuprofen (i.e., the racemic mixture) and Ibuprofen (S)(+), after
one hour dosing, based on the antagonizing property of
Acetylcholine induced writhes in Albino mice.
[0031] FIG. 2 graphically compares the efficacy of L-serine ester
of (.+-.) Ibuprofen, (F1), L-threonine ester of, (I) Ibuprofen
(F2), L-hydroxyproline ester of (.+-.) Ibuprofen (F3),
.+-.Ibuprofen and S(+) Ibuprofen after 3 hour dosing, based on the
antagonizing property of Acetylcholine induced writhes in albino
mice.
[0032] FIG. 3 depicts graphically the dose response relationship to
mean clotting time (MCT) in minutes for the L-serine ester of
acetylsalicylic acid (Formulation 1).
[0033] FIG. 4 depicts graphically the dose response relationship to
mean clotting time (MCT) minutes for the L-hydroxyproline ester of
acetylsalicylic acid (Formulation 2).
[0034] FIG. 5 depicts the dose response relationship to mean
clotting time (MCT) in minutes for the L-threonine ester of
acetylsalicylic acid (Formulation 3)
[0035] FIG. 6 depicts the dose response relationship to mean
clotting time (MCT) in minutes for control (acetylsalicylic
acid).
[0036] FIG. 7 graphically compares the relative efficacy of
L-serine (ester of acetyl salicyclic acid (F.1), L-threonine ester
of acetyl salicylic acid (F.2), L-hydroxyproline ester of
acetylsalicylic acid (F.3), and acetylsalicylic acid (PC) as a
function of mean clotting time in minutes.
[0037] FIG. 8 compares graphically the average clotting time in
minutes of Acetyl salicyclic Acid L-Threonine Ester and BAYER
Asiprin at 325 mg and 81 mg.
[0038] FIG. 9 compares in a bargraph the clotting time in minutes
after 5 day administration of 81 mg Acetyl Salicyclic Acid
L-Threonic Ester and Bayer Aspirin in 3 human volunteers, in which
two volunteers took the Acetyl Salicyclic Acid L-Threonine Ester
and the third took one took just the Bayer Aspirin.
[0039] FIG. 10 compares graphically the percentage increase in
clotting time after a 5 day administration to a patient of
Acetylsalicyclic Acid L-Threonine Ester relative to Aspirin (Bayer)
at 81 mg dose. The plot was derived from the data in Table 29. Two
patients took Acetylsalicyclic Acid L-Threonine Ester b over a
period of five days. The third volunteer took Bayer Aspirin for 5
days.
[0040] FIG. 11 plots the concentration of Aspirin versus time in
four volunteers who took acetyl salicylic Acid L-Threonine Ester
and two volunteers who took Bayer Aspirin.
[0041] FIG. 12 is a plot of the plasma concentration of salicyclic
acid in humans plasma with respect to 4 of volunteers who were
administered Acetyl-salicuclic Acid L-Threonie Ester and two
volunteer who were administered Aspirin.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0042] As used here, the terms "drug", "medicament", and
"pharmaceutical" and "active agent" are being used interchangeably
and refer to the active compound that is administered to the
patient without attachment of the amino acid thereto. Moreover, as
used herein, the drug contains a functional group thereon capable
of reacting with the amino acid, such as NH.sub.2, OH, COOH or
acylating derivatives thereof (e.g., ester, anhydride, amide, and
the like) and the like. When the drug is linked to an amino acid,
the term "amino acid derivative" or "derivative of the present
invention" or synonym thereto is utilized.
[0043] A wide variety of therapeutically active agents can be used
with the present invention. Examples include antacids;
anti-inflammatory substances; coronary dilators; cerebaldilators;
peripheral vasodilators; anti-invectives; psychotropics;
anti-manics; stimulants; anti-histamines; laxatives; decongestants;
vitamins; gastrointestinal sedatives; anti-diarreal preparations;
anti-anginal drugs; vasodilators; anti-arrhythmics;
anti-hypertensive drugs; analgesics; anti-pyretics; hypnotics;
sedatives; anti-emetics; anti-nauseants; anti-convulsants;
neuromuscular drugs; hyper- and hypoglycemic agents; thyroid and
anti-thyroid preparations; diuretics, anti-spasmodics; uterine
relaxants; mineral and nutritional additives; anti-obesity drugs;
anabolic drugs; erythropoietic drugs; anti-asthmatics;
bronchodilators; expectorants; cough suppressants; mucolytics
anti-uricemic drugs; drugs useful for inhibiting organ transplant
rejection; drugs useful for treatment rheumatoid arthritis;
antiviral agents; antibiotics; drugs useful for treating asthma;
drugs useful for treating urinary spasms or infections; calcium
channel blockers; drugs useful for treating malaria; drugs useful
for treating skin diseases; anti-hyperlipidemics; drugs useful for
treating psychological diseases, such as, attention deficient
disorders, schizophrenia, and other psychoses, and the like; drugs
useful for treating tuberculosis; antidiabetics; anti cancer drugs;
drugs useful for treating gastric hyperacidity; drugs useful for
treating eye diseases; and the like. However, the drug utilized
must be capable of bonding to the amino acid. Consquently, it must
have a hydroxy group, an amino group or a carboxy group or an
acylating derivative thereof. By acylating derivative it is meant
that the drug contains a carboxy group or a carboxylic acid
derivative thereof, which has an acyl group thereon, and which can
be hydrolyzed to the acid or salt thereof, such as an amide, ester,
anhydride, and the like.
[0044] The amino acids useful for reacting with the drugs are those
containing the free amino and/or carboxylic acid groups present in
all conventional amino acids. The preferred amino acids are
described in more detail hereinbelow. They are preferably
.alpha.-amino acids. In addition, they are preferably L-amino acids
and more preferably L-.alpha.-amino acids as described hereinbelow.
Of those, some preferred embodiments include those amino acids
having relatively high solubility in aqueous media, for example, in
deionized water at unbuffered aqueous solution at 25.degree. C., of
at least 100 g/L, and more preferably, at least 250 g/L, and even
more preferably at least 500 g/L. For example, glycine and proline
have solubilities in deionized water at 25.degree. C. of
approximately 250 g/L and 1620 g/L, respectively, other preferred
amino acids contain basic amino side chains, such as lysine. For
example, lysine has solubility in deionized water at 25.degree. C.
of approximately 700 g/L. Other preferred amino acids are those
containing hydroxyl side chains, such as hydroxyproline, serine,
and threonine. For example, threonine, hydroxyproline and serine
have solubilities in deionized water at 25.degree. C. of
approximately 100 g/L, 369 g/L and 420 g/L, respectively. Other
preferred embodiments include those amino acids with relatively low
solubility in aqueous media, for example, in deionized water at
25.degree. C. of at most 10 g/L, or for example, at most 2 g/L, or
for example at most 0.6 g/L. For example, the solubility of
tyrosine in deionized water at 25.degree. C. is approximately 0.5
g/L. Such derivatives could be used to produce formulations with
extended release characteristics, due to the limited solubility of
the derivatives.
[0045] Additional preferred amino acids are those containing
carboxylic acid side chains, such as glutamic acid and aspartic
acid. However, non-essential amino acids, and the non-naturally
occurring amino acids can also be bonded to the drug, in accordance
with the present invention.
[0046] The following reaction schemes depict the reactions
discussed hereinabove with respect to the of hydroxyl, carboxyl and
amine containing drugs with various amino acids. In the schemes
below, R is the drug less the functional OH, COOH or NH.sub.2 group
whichever is present, and R.sub.1 is ##STR3## wherein R.sub.0 is
the side chain of the amino acid listed hereinbelow: Reaction
Scheme A: Where the hydroxyl group of the drug is reacted with the
carboxyl group of an amino acid to form the ester derivative
##STR4## Reaction Scheme B: Where the carboxyl group of the drug is
reacted with the hydroxyl group of a hydroxy amino acid wherein the
hydroxy group is on the side chain to form the ester derivative.
##STR5## Reaction Scheme C: Where the amino group of the drug is
reacted with the carboxyl group of the amino acid to from the amide
derivative ##STR6## Reaction Scheme D: Where the carboxyl group of
the drug is reacted with the carboxyl group of the amino acid to
form the anhydride derivative. ##STR7## Reaction Scheme E: Where
the amino group of the drug is reacted with the amino group of the
amino acid to form the azo derivative derivative. ##STR8## Reaction
Scheme F: Where the carboxyl group of the drug is reacted with
amino group of the amino acid to form the amide derivative.
##STR9##
[0047] In the above schemes A-F, the preferred amino acids used are
shown hereinbelow: ##STR10## ##STR11## ##STR12## ##STR13##
[0048] As used herein the term "amino acid" refers to an organic
compound having therein a carboxyl group (COOH) and an amino group
(NH.sub.2) or salts thereof. In solution, at neutral pH, these two
terminal groups, ionize to form a double ionized, through overall
neutral entity identified as zwitterions. The amine donates an
electron to the carboxyl group and the ionic ends are stabilized in
aqueous solution by polar water molecules.
[0049] As used herein, "AA" refers to an amino acid residue, i.e.,
the amino acid without either a hydrogen atom or hydroxy group or
amino group, depending on the linkage to the drug. For example,
"OAA" refers to the amino acid forming an ester linkage with the
oxygen atom of the drug. Thus, in this case, the amino acid is
without a hydroxy group. However, the designation includes the
ester being formed from the bond between the carboxy group of the
amino acid with the hydroxy group of the drug. However, if the
amino acid has a carboxy group on the side chain, such as glutamic
acid or aspartic acid, this also refers to the ester being fromed
from the carboxy group on the side chain and the hydroxy group of
the drug. When aspartic or glutamic acid is the amino acid, the
term OAA refers to an ester linkage between the drug and the amino
acid from the carboxy group on the main chain or on the side chain.
Alternatively, OAA refers to the amino acid forming an ester
linkage from the oxygen atom of the side chain of a hydroxy
containing amino acid, and the carboxylic acid functionality of the
drug. However, the definition should be made clear by the context.
For example, the nomenclature, especially in the claims, is
accompanied by language reciting that the bond is being formed from
a group, such as amino or OH group of the amino acid. Thus, for
example, if the term is OAA, and if it is meant to refer to a ester
bond between the OH group on the side chain and the acyl group of
the drug, the term would be accompanied by a statement that the
amino acid is without the hydroxy group of the side chain and that
the ester is formed via the hydroxy on the side chain.
Alternatively, if the OAA refers to the ester linkage between the
carboxy group of the amino acid and the oxygen atom of the drug,
the term should be accompanied by a statement that the amino acid
is without the hydroxy group and that the ester linkage is through
the acyl group of the amino acid. Further, if the structure of the
drug to which the amino acid is bonded is identified or provided,
the meaning will be apparent to the skilled artisan without
explanation.
[0050] The term NHAA refers to an amino acid residue (an amino acid
less an OH group on the carboxy group) bonded through its acyl
group to the amino group of the drug, thereby forming an amide
linkage therebetween. Further, it should be noted that if the side
chain of the amino acid has a carboxy group thereon, another amide
bond may occur linking the carboxy group of the side chain and the
amino group of the drug. Thus, the NHAA refers to the amide linkage
between a carboxy group on the amino acid and the amino group on
the drug. Thus, the term NHAA refers to the amide bond formed
between the carboxy group of the drug and the amino group of the
amino acid, either in the main chain or on the side chain.
Alternatively, it can refer to the amide linkage between the amino
group of the amino acid and the carboxy group or acylating
derivative of the drug. Again, the term will be accompanied by
language in the claims to identify the meaning, if it is not clear
from context. However, if the structure is given, the meaning will
be apparent to the skilled artisan without explanation.
[0051] If there is a bond between the hydroxy group on the side
chain of the amino acid, such as hydroxyproline, theonine,
tyrosine, serine and the like, then the nomerclature C(O) AA will
be utilized, signifying that the linkage is between the carboxy
group on the drug and the OH group on the amino acid side
chain.
[0052] The term ##STR14## is used to denote that an acid anhydride
is formed from the reaction of the carboxyl group of the drug and
the carboxyl group of the amino acid, whether it be on the main
chain or side chain of the amino acid, to form an carboxylic acid
anhydride.
[0053] The term ##STR15## is used to denote the azo bond formed
from the reaction of the amino group from the drug and the amino
group of the amino acid.
[0054] It is the side groups that distinguish the amino acids from
each other. Some amino acids, such as lysine, have amino groups on
the side chain; other amino acids have side chains containing
hydroxy groups, such as threonine, serine, hydroxyproline, and
tyrosine; some amino acids have carboxy groups on the side chain,
such as glutamic acid or aspartic acid. These functional groups on
the side chain also can form a covalent bond with the drug, such as
esters, amides, and the like. When these side groups become
involved in these linkages, such as hydroxy group, the bond may be
depicted as OAA, wherein AA is an amino acid residue having a side
chain with a hydroxy group, but without the hydroxy group. Thus, AA
by this definition, refers to the amino acid without the hydroxy
side group since the hydroxy moiety took part in the reaction in
forming the ester. Moreover, when an ester is formed between the
hydroxy group of the amino acid and the carboxy group of the drug,
the hydroxy group on the carboxy group forms a byproduct with the
hydrogen of the hydroxy group, thus, the resulting product does not
have the OH group on the carboxy group, but just the acyl moiety.
When the bond is depicted as C(.dbd.O)--NHAA, this means that the
amino acid forms as an amide bond between the carboxy group on the
drug and the amino group of the amino acid. However, as written,
since the NH from the amide bond comes from the amino acid, AA is
the amino acid without the amino group.
[0055] The preferred amino acids are the naturally occurring amino
acids. It is more preferred that the amino acids are the
.alpha.-amino acids. It is also preferred that the amino acids are
in the L-configuration. The preferred amino acids include the
twenty essential amino acids. The preferred amino acids are Lysine
(Lys), Leucine (Leu), Isoleucine (Ile), Glycine (Gly), Aspartic
Acid (Asp), Glutamic Acid (Glu), Methionine (Met), Alanine (Ala),
Valine (Val), Proline (Pro), Histidine (His), Tyrosine (Tyr),
Serine (Ser), Norleucine (Nor), Arginine (Arg), Phenylalanine
(Phe), Tryptophan (Trp), Hydroxyproline (Hyp), Homoserine (Hsr),
Carnitine (Car), Ornithine (Ort), Canavanine (Cav), Asparagine
(Asn), Glutamine (Gln), Camosine (Can), Taurine (Tau), djenkolic
Acid (Djk), .gamma.-aminobutyric Acid (GABA), Cysteine (Cys)
Cystine (Dcy), Sarcosine (Sar), Threonine (Thr) and the like. The
even more preferred amino acids are the twenty essential amino
acids, Lys, Leu, Ile, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr,
Thr, Arg, Phe, Trp, Gln, Asn, Cys and Ser. The most preferred amino
acids are those having a hydroxy group on the side chain,
preferably Hyp, Thr, Ser and Tyr and most preferably L-Hyp, L-Tyr,
L-Ser and L-Thr. The most preferred amino acids are L-Hyp and
L-Thr, and especially L-Thr.
[0056] The derivatives are prepared from a drug having a group
thereon which can react with the amino acid.
[0057] The preferred drugs that are reacted with amino acids in
accordance with various schemes are as follows. The drugs and their
reaction schemes stated here are not exhaustive, and are typical
examples only. TABLE-US-00001 Reaction Schemes Drug A B C D E F
Abacavir YES YES YES Acarbose YES Acebutolol YES YES Adefovir YES
Albuterol YES YES Amlodipine.sup.1 YES Amphotericin B YES YES YES
YES Amprenavir YES YES YES Atenolol YES YES YES Atorvastatin YES
YES YES YES Atropine YES Baclofen YES YES YES YES YES Benazeprilat
YES YES YES YES Betaxolol YES YES Bicalutamide YES YES Biotin YES
YES YES YES Biperiden YES Bisoprolol YES YES Bitolterol YES YES
Brinzolamide YES YES Bupivacaine YES Buprenorphine YES Bupropion
YES Butorphanol YES Capacitabine YES Captopril YES YES YES YES
Carbidopa YES YES YES YES YES YES Carnitine YES YES YES YES
Carteolol YES YES Cefditoren YES YES YES YES YES Cerivastatin YES
YES YES YES Chloramphenicol YES Cisapride YES YES Clopidogrel Acid
YES YES YES Clorazepic Acid YES YES YES YES Cycloserine YES YES
Cytarabine YES YES YES Danazol YES Dextroamphetamine YES YES
Diclofenac YES YES YES YES Didanosine YES YES Digoxin YES
Divalproex YES YES YES Docetaxel YES YES Dorzolamide YES YES
Dyphylline YES Dysopyramide YES YES Efavirenz YES Enalaprilat YES
YES YES YES Ephedrine YES YES Eplerenone YES YES YES Eprosartan YES
YES YES Esmolol YES YES Estramustine YES Ethambutol YES YES
Ethchlorvynol YES Ethosuximide YES Ethotoin YES Etidocaine YES
Etoposide YES Ezetimibe YES Fenofibrate YES YES YES Fenoprofen YES
YES YES Fexofenadine YES YES YES YES Finasteride YES Fluoxetine YES
Fluticasone YES Fluvastatin YES YES YES YES Folic Acid YES YES YES
YES YES Fosinoprilat YES YES Frovatriptan YES YES Fulvestrant YES
Gabapentin* YES YES YES YES YES Ganciclovir YES Glimepiride YES
Goserelin YES Hydroxychloroquine YES Hydroxyzine YES Hyoscyamine
YES Ibuprofen YES YES YES Ibutilide YES YES Indapamide YES YES
Indinavir YES YES Ipratropium YES Irinotecan YES Isosorbide YES
Isradipine.sup.5 YES Ketoprofen YES YES YES Ketorolac YES YES YES
Labetalol YES YES Lamivudine YES YES YES Lamivudine YES YES YES
Lansoprazole YES Latanoprost Acid YES YES YES YES Leuprolide YES
Levobunolol YES YES YES Levodopa YES YES YES YES YES YES
Levorphanol YES Liothyronine YES YES YES YES YES YES Lisinopril YES
YES YES YES YES Lopinavir YES YES Lorazepam YES Lovastatin YES YES
YES YES Medroxyprogesterone YES Mefloquine YES YES Megestrol YES
Mephobarbital YES Mepivacaine YES Metaproterenol YES YES Metformin
YES YES Methamphetamine YES Methohexital YES Methotrexate YES YES
YES Methylphenidate YES YES YES YES Methylphenidate.sup.6 YES
Methylprednisolone YES Metolazone YES YES Metoprolol YES YES
Mexiletine YES YES Miglitol YES Miglitol YES Moexiprilat YES YES
YES YES Mometasone YES Montelukast YES YES YES YES Nadolol YES YES
Nalbuphine YES Naproxen YES YES YES Naratriptan YES YES Nateglinide
YES YES YES YES Nelfinavir YES YES Niacin YES YES YES
Nicardipine.sup.3 YES Nimidipine.sup.4 YES Nisoldipine.sup.2 YES
Norgestimate YES Octreotide YES YES Ofloxacin YES YES YES
Olmesartan YES YES YES Omeprazole YES YES Paclitaxel YES YES
Pantothenic Acid YES YES YES YES YES Paroxetine YES YES Paroxetine
YES Pemoline YES YES Penbutolol YES YES Penicillamine YES YES YES
YES YES Pentazocine YES Pentobarbital YES Perindoprilat YES YES YES
YES Phenylephrine YES YES Phenylpropanolamine YES YES YES Pindolol
YES YES Pioglitazone YES Pirbuterol YES Pramipexole YES YES
Pravastatin YES YES YES YES Propafenone YES YES Propranolol YES YES
Pseudoephedrine YES YES Quinacrine YES Quinaprilat YES YES YES YES
Quinethazone YES YES Quinidine YES Quinine YES Ramiprilat YES YES
YES YES Reboxetine YES Repaglinide YES YES YES YES Repaglinide YES
YES YES YES YES Ribavirin YES YES YES Ritonavir YES YES Ropivacaine
YES Rosiglitazone YES Rosuvastatin YES YES YES YES Salmeterol YES
YES Sertraline YES Simavastatin YES YES YES YES Sirolimus YES
Sotalol YES YES Sulfa Drugs YES YES Sulfasalazine YES Sumitriptan
YES YES Tacrolimus YES Tazorotene YES YES YES Telmesartan YES YES
YES Tenofovir YES Terbutaline YES YES Thyroxine YES YES YES YES YES
Tiagabine YES YES YES Timolol YES YES Tirofiban YES YES YES YES
Tocainide YES YES Tramadol YES Trandolaprilat YES YES YES YES
Tranylcypromine YES YES Treprostinil YES YES YES YES Triamcinolone
YES Troglitazone YES YES Unoprostone YES YES YES Valsartan YES YES
YES Venlafaxine YES Vidarabine YES YES YES Warfarin YES Zalcitabine
YES YES YES Zidovudine YES YES Zolmitriptan YES YES .sup.1In case
of Amlodipine, one can replace 5-methyl ester moiety with an amino
acid resulting in better therapeutic index. In case of intact
Amlodipine molecule, biotransformation results in generation of
methanol due to solvolysis of 5-methyl ester, which is highly
toxic, and replacement of this with a non-toxic naturally occurring
amino acid with much less toxicity. In addition, the amino acid can
form an amide bond # with the primary and secondary amine groups on
the Amlodipine. Moreover, an amino acid can form an amide or ester
bond at the 3 position of the ring. This same argument goes for
rest of the products in this category stated below: .sup.2In case
of Nisoldipine, replace 5-methyl ester with an amino acid for
better therapeutic index, and no loss of activity. Also an amide
linkage may form between the amino group of the Nisoldipine and the
amino acid. In addition, an amide linkage or ester linkage can form
between the 3 isobutyl ester and the amino acid. .sup.3For
Nicardipine, replace 5-methyl ester with an amino acid for better
therapeutic index. In addition, the ester at the 3-position of the
ring can form an ester linkage with the OH group on the side chain.
Further, the amino group can form an amide bond with a carboxy
group or acylating derivative thereof of the amino acid. .sup.4For
Nimodipine, one could replace 5 (1-methyl)ethyl ester with an amino
acid for better therapeutic index. In addition, an amide can form
between the amine group of the ring and the amino acid. In
addition, the 3 position of Nimodipine can form an amide bond or an
ester bond with an amino acid. .sup.5For Isradipine, replace 5
methyl ester with an amino acid. The methyl ester is the active,
and apparently the carboxylic acid derivative is not active.
.sup.6An amide linkage can be formed between the secondary nitrogen
atom in the ring and an amino acid. Moreover, the 3-position of the
ring can form an amide or ester linkage with an amino acid.
Replacing the methoxy group with amino acid will still maintain
activity, but none of the toxicity of methylphenidate. In addition,
an amide bond can form between the nitrogen atom of the pyridine
and the amino acids.
[0058] The amino acid derivative of the present invention contains
amino groups and as such are basic in nature. They are capable of
forming a wide variety of pharmaceutically acceptable salts with
various inorganic and organic acids. These acids that may be used
to prepare pharmaceutically acceptable acid addition salts of such
basic compounds are those that form non-toxic acid addition salts,
i.e., salts containing pharmaceutically acceptable anions, such as
the hydrochloride, hydrobromide, hydroiodide, nitride, sulfate,
bisulfate, phosphate, formate, acetate, citrate, tartate, lactate,
and the like. The amino acid derivative of the present invention
can form pharmaceutically acceptable salts with acids.
[0059] As indicated herein, in one embodiment, the present
invention is directed to a derivative wherein the derivative
comprises a drug, e.g., cyclosporine of the formula ##STR16## and
an amino acid esterified to the MeBmt (x-y=CH.dbd.CH) or dihydro
MeBmt portion, (wherein x-y=CH.sub.2CH.sub.2). The amino acid is
attached to the cyclosporine and to the other other drugs by a
covalent bond.
[0060] The compounds of the present invention are prepared by art
recognized techniques. For example, if the drug contains an OH
group, said as cyclosporin, then an amino acid or an acylating
derivatives thereof, such as the acid halide, e.g., amino acid
fluoride, amino acid chloride, or an amino acid alkyl ester wherein
alkyl group contains 1-6 carbon atoms is reacted with the carboxy
group of the drug, e.g., cyclosporine under esterification
condition. Preferably, the reaction is conducted in the presence of
an acid, such as hydrochloric acid, hydrobromic acid,
p-toluenesulfonic acid and the like. Alternatively, as described
hereinabove, if the drug has an amino group thereon, then the amino
acid may be reacted with the drug under amide forming conditions to
form an amide as the covalent bond. Or if the drug has a carboxy
group or acylating derivative thereon, it may be reacted with the
amino group of the amino acid to form an amide under amide forming
conditions to form an amide bond between the amino acid and the
drug. Additionally if the drug has a carboxy group thereon, the
hydroxy group of the side chain of the amino acid may be reacted
with the carboxy group or acylating derivative thereon under
esterification conditions to form the ester linkage between the
amino acid and the drug, as described hereinabove.
[0061] If the amino acid has a group thereon which is reactive
under the reaction conditions it is protected by a protecting group
known in the art. After the completion of the reaction, the
protecting group is removed. Examples of protecting groups that
could be used are described in the book entitled, "Protective Group
in Organic Synthesis" by Theodora W. Greene, John Wiley & Sons,
1981, the contents of which are incorporated by reference.
[0062] For example, if amino acids with carboxylic groups in their
side chains, for example, aspartic acid and glutamic acid, are used
in the aforementioned syntheses, these will generally require
protection of the side chain carboxylic acid. Suitable protecting
groups can be esters, such as cyclohexyl esters, t-butyl esters,
benzyl esters, allyl esters, 9-fluorophenyl-methyl groups or
adamantyl groups, such as 1- or 2-adamantyl which can be removed
after the esterfication reaction is completed using techniques
known to one of ordinary skill in the art.
[0063] If amino acids with hydroxyl groups in their side chains,
for example, serine, threonine, hydroxyproline, and the like and
amino acids with phenolic groups in their side chains, for example,
tyrosine, and the like are used in the aforementioned
esterification reactions, they will desirably require protection of
the chain hydroxyl or phenolic group. Suitable protecting groups
for the hydroxyl side chain groups can be ethers, such as benzyl
ether or t-butyl ether and the like. Removal of the benzyl ether
can be effected by liquid hydrogen fluoride, while the t-butyl
ether can be removed by treatment with trifluoroacetic acid.
Suitable protecting groups for the phenolic side chain groups can
be ethers, as above, including benzyl or t-butyl ether or
2,6-dichlorobenzyl, 2-bromobenzyloxycarbonyl, 2,4-dintrophenyl and
the like.
[0064] Moreover, the products can be purified to be made
substantially pure by techniques known to one of ordinary skill in
the art, such as by chromatography, e.g., HPLC, crystallization and
the like. By substantially "pure" it is meant that the product
contains no more than about 10% impurity therein.
[0065] The amino acid derivatives of the present invention
encompass pharmaceutical acceptable salts, pharmaceutical
acceptable solvates, enantiomers, diastereomers, N-Oxides, and
polymorphs thereof, as described herein, and they can be associated
along with a pharmaceutical acceptable carrier, and optionally but
desirably pharmaceutically acceptable excipients and made into
pharmaceutical composition using techniques known to one of
ordinary skill in the art.
[0066] All the various stereoisomers of the amino acid derivatives,
such as enantiomers and dieastercomers are comtemplated to be
within the scope of the present invention. However, if the
preferred drug has a preferred steroisomeric form, it is also
preferred in the present invention. Moreover, the preferred amino
acid derivatives of the present invention has the asymmetric carbon
atom on the group adjacent to the amino and carboxy group from the
amino acid moiety in the L configuration.
[0067] The amino acid derivatives described herein have the same
utility as that drug without the amino acid moiety bonded thereto.
The amino acid derivatives are used in therapeutically effective
amounts.
[0068] The physician will determine the dosage of the derivatives
of the present invention which will be most suitable and it will
vary with the form of administration and the particular compound
chosen, and furthermore, it will vary depending upon various
factors, including but not limited to the patient under treatment
and the age of the patient, the severity of the condition being
treated and the like and the identify of the derivative
administered. He will generally wish to initiate treatment with
small dosages, substantially less than the optimum dose of the
compound, and increase the dosage by small increments until the
optimum effect under the circumstances is reached. It will
generally be found that when the composition is administered
orally, larger quantities of the compounds of the present invention
will be required to produce the same effect as a smaller quantity
given parenterally. The amino acid derivatives of the present
invention have the same utility as the corresponding drug in the
non amino acid derivatized form and the dosage level is generally
no greater than that is generally employed with these other
therapeutic agents. When given parenterally, the compounds are
administered generally in dosages of, for example, about 0.001 to
about 10,000 mg/kg/day, also depending upon the host and the
severity of the condition being treated and the compound
utilized.
[0069] In a preferred embodiment, the compounds of the present
invention utilized are orally administered in amounts ranging from
about 0.01 mg to about 1000 mg per kilogram of body weight per day,
depending upon the particular mammalian host or the disease to be
treated, more preferably from about 0.1 to about 500 mg/kg body
weight per day. This dosage regimen may be adjusted by the
physician to provide the optimum therapeutic response. For example,
several divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation.
[0070] The amino acid derivative of the present invention may be
administered in any convenient manner, such as by oral,
intravenous, intramuscular or subcutaneous routes.
[0071] The amino acid derivative of the present invention may be
orally administered, for example, with an inert diluent or with an
assimilable edible carrier, or it may be enclosed in hard or soft
shell gelatin capsules, or it may be compressed into tablets, or it
may be incorporated directly into the food of the diet. For oral
therapeutic administration, the amino acid derivative of the
present invention may be incorporated with excipients and used in
the form of ingestible tablets, buccal tablets, troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 1% of the
derivative. The percentage of the compositions and preparations
may, of course, be varied and may conveniently be between about 5
to about 80% of the weight of the unit. The amount of the amino
acid derivative used in such therapeutic compositions is such that
a suitable dosage will be obtained. Preferred compositions or
preparations according to the present invention contain between
about 200 mg and about 4000 mg of amino acid derivative. The
tablets, troches, pills, capsules and the like may also contain the
following: A binder such as gum tragacanth, acacia, corn starch or
gelatin; excipients such as dicalcium phosphate; a disintegrating
agent such as corn starch, potato starch, alginic acid and the
like; a lubricant such as magnesium stearate; and a sweetening
agent such as sucrose, lactose or saccharin may be added or a
flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring. When the dosage unit form is a capsule, it may contain,
in addition to materials of the above type, a liquid carrier.
[0072] Various other materials may be present as coatings or
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup or elixir may contain the active compound,
sucrose as a sweetening agent, methyl and propylparabens as
preservatives, a dye and flavoring such as cherry or orange flavor.
Of course, any material used in preparing any dosage unit form
should be pharmaceutically pure and substantially non-toxic in the
amounts employed. In addition, the active compound may be
incorporated into sustained-release preparations and formulations.
For example, sustained release dosage forms are contemplated
wherein the active ingredient is bound to an ion exchange resin
which, optionally, can be coated with a diffusion barrier coating
to modify the release properties of the resin or wherein the
derivative of the present invention is associated with a sustained
release polymer known in the art, such as
hydroxypropylmethylcellulose and the like.
[0073] The amino acid derivative may also be administered
parenterally or intraperitoneally. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols, e.g., PEG 100,
PEG 200, PEG 300, PEG 400, and the like, and mixtures thereof and
in oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0074] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases the form is
usually sterile and must be fluid to the extent that syringability
exists. It must be stable under the conditions of manufacture and
storage and usually must be preserved against the contaminating
action of microorganisms such as bacteria and fungi. The carrier
can be a solvent or dispersion medium containing, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol,
and one or more liquid polyethylene glycol, e.g. as disclosed
herein and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersions and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0075] Sterile injectable solutions are prepared by incorporating
the amino acid derivative of the present invention in the required
amount in the appropriate solvent with various of the other
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the various sterilized amino acid derivative of the present
invention into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders, the above
solutions are vacuum dried or freeze-dried, as necessary.
[0076] The amino acid derivative of the present invention can also
be applied topically, as e.g., through a patch using techniques
known to one of ordinary skill in the art.
[0077] The amino acid derivative of the present invention can be
administered buccally by preparing a suitable formulation of the
derivative of the present invention and utilizing procedures well
known to those skilled in the art. These formulations are prepared
with suitable non-toxic pharmaceutically acceptable ingredients.
These ingredients are known to those skilled in the preparation of
buccal dosage forms. Some of these ingredients can be found in
Remington's Pharmaceutical Sciences, 17.sup.th edition, 1985, a
standard reference in the field. The choice of suitable carriers is
highly dependent upon the exact nature of the buccal dosage form
desired, e.g., tablets, lozenges, gels, patches and the like. All
of these buccal dosage forms are contemplated to be within the
scope of the present invention and they are formulated in a
conventional manner.
[0078] The formulation of the pharmaceutical compositions may be
prepared using conventional methods using one or more
physiologically and/or pharmaceutically acceptable carriers or
excipients. Thus, the amino acid derivatives and their
pharmaceutically acceptable salts and solvates may be formulated
for administration by inhalation or insufflation (either through
the mouth or the nose) or oral, buccal, parenteral, or rectal
administration. For oral administration, the pharmaceutical
compositions may take the form of, for example, tablets or capsules
prepared by conventional means with pharmaceutically acceptable
excipients such as binding agents (for example, pregelatinized
maize starch, polyvinylpyrrolidone, or hydroxypropylmethyl
cellulose); fillers (for example, lactose, microcrystalline
cellulose or calcium hydrogen phosphate); lubricants (for example,
magnesium stearate, talc, or silica); disintegrants (for example,
potato starch, or sodium starch glycolate); or wetting agents (for
example, sodium lauryl sulfate). The tablets may be coated by
methods well known in the art.
[0079] Liquid preparations for oral administration may take the
form of, for example, solutions, syrups, or suspensions, or they
may be presented as a dry product for constitution with water or
other suitable vehicles before use. Such liquid preparations may be
prepared by conventional means with pharmaceutically acceptable
additives, such as suspending agents (for example, sorbitol syrup,
corn syrup, cellulose derivatives or hydrogenated edible oils and
fats); emulsifying agents (for example, lecithin or acacia);
non-aqueous vehicles (for example, almond oil, oily esters, ethyl
alcohol or fractionated vegetable oils); and preservatives (for
example, methyl or propyl p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring and
sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated to give controlled
release of the amino acid derivative of the present invention.
[0080] The amino acid derivative of the present invention may be
formulated for parenteral administration by injection, for example,
by bolus injection or continuous infusion. Formulations for
injection may be presented in unit dosage form, for example, in
ampoules, or in multi-dose containers, with an added preservative.
The compositions may take such forms as suspension, solutions or
emulsions in oily or aqueous vehicles, and may contain formulation
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the amino acid derivative may be in the powder form
for constitution with a suitable vehicle, for example, sterile
pyrogen-free water, before use.
[0081] The amino acid derivatives of the present invention may also
be formulated in rectal compositions such as suppositories or
retention enemas, for example, containing conventional suppository
bases such as cocoa butter or other glycerides.
[0082] In addition to the formulations described previously, the
amino acid derivative of the present invention may also be
formulated as a depot preparation. Such long acting formulations
may be administered by implantation (for example, subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the amino acid derivatives may be formulated with suitable
polymeric or hydrophobic materials (for example, as an emulsion in
an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
[0083] The pharmaceutical compositions containing the amino acid
derivatives of the present invention may, if desired, be presented
in a pack or dispenser device which may contain one or more unit
dosage forms containing the active ingredients. The pack may for
example comprise metal or plastic foil, such as blister pack. The
pack or dispenser device may be accompanied by instructions for
administration.
[0084] In tablet form, it is desirable to include a lubricant which
facilitates the process of manufacturing the dosage units;
lubricants may also optimize erosion rate and drug flux. If a
lubricant is present, it will be present on the order of 0.01 wt. %
to about 2 wt. %, preferably about 0.01 wt. % to 0.5 wt, %, of the
dosage unit. Suitable lubricants include, but are not limited to,
magnesium stearate, calcium stearate, stearic acid, sodium
stearylfumarate, talc, hydrogenated vegetable oils and polyethylene
glycol. As will be appreciated by those skilled in the art,
however, modulating the particle size of the components in the
dosage unit and/or the density of the unit can provide a similar
effect--i.e., improved manufacturability and optimization of
erosion rate and drug flux--without addition of a lubricant.
[0085] Other components may also optionally be incorporated into
the dosage unit. Such additional optional components include, for
example, one or more disintegrants, diluents, binders, enhancers,
or the like. Examples of disintegrants that may be used include,
but are not limited to, crosslinked polyvinylpyrrolidones, such as
crospovidone (e.g., Polyplasdone.RTM. XL, which may be obtained
from GAF), cross-linked carboxylic methylcelluloses, such as
croscanmelose (e.g., Ac-di-sol.RTM., which may be obtained from
FMC), alginic acid, and sodium carboxymethyl starches (e.g.,
Explotab.RTM., which may be obtained from Edward Medell Co., Inc.),
agar bentonite and alginic acid. Suitable diluents are those which
are generally useful in pharmaceutical formulations prepared using
compression techniques, e.g., dicalcium phosphate dihydrate (e.g.,
Di-Tab.RTM., which may be obtained from Stauffer), sugars that have
been processed by crystallization with dextrin (e.g.,
co-crystallized sucrose and dextrin such as Di-Pak.RTM., which may
be obtained from Amstar), calcium phosphate, cellulose, kaolin,
mannitol, sodium chloride, dry starch, powdered sugar and the like.
Binders, if used, are those that enhance adhesion. Examples of such
binders include, but are not limited to, starch, gelatin and sugars
such as sucrose, dextrose, molasses, and lactose. Permeation
enhancers may also be present in the novel dosage units in order to
increase the rate at which the active agents pass through the
buccal mucosa. Examples of permeation enhancers include, but are
not limited to, dimethylsulfoxide ("DMSO"), dimethyl formamide
("DMF"), N,N-dimethylacetamide ("DMA"), decylmethylsulfoxide
("C.sub.10MSO"), polyethylene glycol monolaurate ("PEGML"),
glycerol monolaurate, lecithin, the 1-substituted
azacycloheptan-2-ones, particularly
1-n-dodecylcyclazacycloheptan-2-one (available under the trademark
Azone.RTM. from Nelson Research & Development Co., Irvine,
Calif.), lower alkanols (e.g., ethanol), SEPA.RTM. (available from
Macrochem Co., Lexington, Mass.), cholic acid, taurocholic acid,
bile salt type enhancers, and surfactants such as Tergitol.RTM.,
Nonoxynol-9.RTM. and TWEEN-80.RTM..
[0086] Flavorings may be optionally included in the various
pharmaceutical formulations. Any suitable flavoring may be used,
e.g., mannitol, lactose or artificial sweeteners such as aspartame.
Coloring agents may be added, although again, such agents are not
required. Examples of coloring agents include any of the water
soluble FD&C dyes, mixtures of the same, or their corresponding
lakes.
[0087] In addition, if desired, the present dosage units may be
formulated with one or more preservatives or bacteriostatic agents,
e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol,
benzalkonium chloride, or the like.
[0088] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents well known in the art that are associated with
drugs, medicaments, or active agents. Except insofar as any
conventional media or agent is incompatible with the amino acid
derivative, the use of these solvents, dispersion media, coatings
and various isotonic and delaying agents and antibacterical and
antifungal agents in the therapeutic compositions of the present
invention is contemplated. Supplementary active ingredients can
also be incorporated into the compositions.
[0089] Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the subjects to be
treated, each unit containing a predetermined quantity of amino
acid derivative calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0090] The amino acid derivative is compounded for convenient and
effective administration in effective amounts with a suitable
pharmaceutically acceptable carrier in dosage unit form as
hereinbefore described. A unit dosage, for example, contains the
amino acid derivative of the present invention in amounts ranging
from about 10 mg e.g. in humans, or as low as 1 mg (for small
animals) to about 2000 mg. If placed in solution, the concentration
of the amino acids derivative preferably ranges from about 10 mg/mL
to about 250 mg/mL. In the case of compositions containing
supplementary active ingredients, the dosages are determined by
reference to the usual dose and manner of administration of the
said ingredients. In the case of buccal administration, the amino
acid derivatives are preferably in the buccal unit dosage form
present in an amount ranging from about 10 to about 50 mg.
[0091] The amino acid derivatives of the present invention are
effective in treating disease or conditions in which the
corresponding drug (without the amino acid derivative of the
present invention) normally are used.
[0092] As used herein the term "treating" refers to reversing,
alleviating or inhibiting the progress of a disease, disorder or
condition, or one or more symptoms of such disease, disorder or
condition, to which such term applies. As used herein, "treating"
may also refer to decreasing the probability or incidence of the
occurrence of a disease, disorder or condition in a mammal as
compared to an untreated control population, or as compared to the
same mammal prior to treatment. For example, as used herein,
"treating" may refer to preventing a disease, disorder or
condition, and may include delaying or preventing the onset of a
disease, disorder or condition, or delaying or preventing the
symptoms associated with a disease, disorder or condition. As used
herein, "treating" may also refer to reducing the severity of a
disease, disorder or condition or symptoms associated with such
disease, disorder or condition prior to a mammal's affliction with
the disease, disorder or condition. Such prevention or reduction of
the severity of a disease, disorder or condition prior to
affliction relates to the administration of the composition of the
present invention, as described herein, to a subject that is not at
the time of administration afflicted with the disease, disorder or
condition. As used herein "treating" may also refer to preventing
the recurrence of a disease, disorder or condition or of one or
more symptoms associated with such disease, disorder or condition.
The terms "treatment" and "therapeutically," as used herein, refer
to the act of treating, as "treating" is defined above.
[0093] Prophylaxis or preventing, or any like term, refers to
decreasing the probability or incidence of the occurrence of a
disease, disorder or condition in a mammal. It also includes
delaying or preventing the onset of a disease, disorder or
condition or delaying the symptoms associated with a disease,
disorder or condition. In addition, it also refers to retarding the
occurrence of a disease, disorder or condition in a mammal.
[0094] As used herein the term "patient" or "subject" refers to a
warm blooded animal, and preferably mammals, such as, for example,
cats, dogs, horses, cows, pigs, mice, rats and primates, including
humans. The preferred patient is humans.
[0095] The amino acid derivatives of the present invention exhibit
the same utility as the corresponding drug without the amino acid
linkage. The amino acid derivative of the present invention
exhibits an enhanced therapeutic quality. That is, they exhibit at
least one and more preferably at least two enhanced therapeutic
qualities relative to the drug which has not been transformed to
the derivative of the present invention prior to administration.
These include, but are not limited to [0096] a. Improved taste,
smell [0097] b. Desired octanol/water partition coefficient (i.e.,
solubility in water/fat)
[0098] The various amino acids have different solubility in aqueous
solutions. By selecting a particular amino acid, the octanol water
partition coefficient can be affected. For example, many drugs in
the following list are highly hydrophobic. The amino acids are
highly hydrophilic. For example, assume propofol is the drug and
lysine is the amino acid. Propofol is completely insoluble in
water, while lysine is soluble to the extent of 700 mg/ml. When
these two diverse molecules are esterified via an ester bond, the
resulting lysine ester of propfol has a solubility in water in
excess of 250 mg/ml.
[0099] On the other hand, cromolyn sodium is highly water soluble.
For all practical purposes, it is not absorbed when administered
orally. By affecting its water solubility one could improve
absorption. In this case, one would look for conditions opposite to
that of propofol, i.e., the goal is to decrease water solubility.
By choosing the appropriate low water soluble amino acids, such as
tyrosine, one can achieve proper hydrophilic/lipophilic balance.
[0100] c. Improved stability in-vitro and in-vivo [0101] d.
Enhanced penetration of blood-brain barrier [0102] e. Elimination
of first-pass effect in liver, i.e., the drug not metabolized in
liver and therefore more drug in system circulation [0103] f.
Reduction of entero-hepatic recirculation (this improves
bio-availability) [0104] g. Painless injections with parenteral
formulations [0105] h. Improved bio-availability [0106] i. Improved
changes in the rate of absorption (increase vs lack thereof) [0107]
j. Reduced side effects [0108] k. Dose proportionality
[0109] A dose proportionality claim requires that when the drug is
administered in escalating doses, proportionally escalating amounts
of active drug is delivered into the blood stream. This is measured
by determining the area under the plasma concentration vs. time
curve obtained after administering a drug via any route other than
IV route and measuring the same in the plasma/blood. A simple
mathematical procedure is as follows:
[0110] For example, a drug is administered at e.g., 3 different
doses, 10, 100 and 1000 mg, orally to a patient; the area under the
plasma concentration time curve (AUC) is measured. Then each total
AUC is divided by the dose, and the result should be the same for
all three doses. If it is the case, then there is dose
proportionality. Lack of dose proportionality indicates any one or
more of the pharmacokinetic/pharmacological mechanisms are
saturable, including absorption, metabolism or the number of
receptor sites available for pharmacological response.
[0111] For example in the above study, assume the AUC values of
100, 1000 and 10,000 are obtained, in this case the dose
proportionality is inappropriate. When there is lack of dose
proportionality, there is either more or less amount of drug in the
plasma, depending upon which mechanism is saturable. The following
are the possibilities: Saturable Absorption. If this is the case,
as the dose is increased, proportionally less and less of the drug
is absorbed, hence overall AUC will decrease as the dose is
increased.
[0112] Saturable metabolism of elimination. If thus is the case,
then more and more of the drug will be circulating in the blood,
and the AUC will increase with increasing dose.
[0113] Saturable pharmacological receptor sites: In this case,
since all the receptor sites will eventually be occupied by the
drug, any additional drug will not increase the response. Thus,
increasing dose will not result in increasing response.
[0114] Dose proportionality is an excellent response profile, since
one can predict accurately the pharmacological response and
curative power at all doses. Thus dose proportionality is a
desirable quality for any drug. Furthermore, achievement of dose
proportionality is also dependent upon the formulation, and
fed/fasted differences. [0115] l. Selective hydrolysis of the
derivative at site of action [0116] m. Controlled release
properties [0117] n. Targeted drug delivery [0118] o. Reduction in
toxicity, hence, improved therapeutic ratio [0119] p. Reduced dose
[0120] q. Alteration of metabolic pathway to deliver more drug at
the site of action [0121] r. Increased solubility in aqueous
solution [0122] s. Enhanced efficacy
[0123] The amino acid derivatives are available in various dosage
forms and they are prepared by conventional methods: [0124] i. Oral
liquid dosage (Controlled release and immediate release liquids
containing sugar and sugar free, dye and dye free, alcohol and
alcohol free formulations, including chewable tablets) [0125] ii.
Oral solid dosage (Controlled release and immediate release
tablets, capsules and caplets [0126] iii. Intravenous (Injections,
both ready to use and lyophilized powders) [0127] iv. Intramuscular
(Injections, both ready to use and lyophilized powders) [0128] v.
Subcutaneous (Injections, both ready to use and lyophilized
powders) [0129] vi. Transdermal (Mainly patches) [0130] vii. Nasal
(Sprays, formulations for nebulizer treatments) [0131] viii.
Topical (Creams, ointments) [0132] ix. Rectal (Creams, ointments
and suppositories) [0133] x. Vaginal (Creams, ointments and
pessaries) [0134] xi. Ocular (Drops and ointments) [0135] xii.
Buccal (Chewable and now chewable tables)
[0136] Many drugs discussed herein, especially in the table
hereinbelow are characteristically highly hydrophobic and readily
precipitate in the presence of even very minor amounts of water,
e.g., on contact with the body (e.g., stomach fluids). It is
accordingly extremely difficult to provide, e.g., oral formulations
which are acceptable to the patient in terms of form and taste,
which are stable on storage and which can be administered on a
regular basis to provide suitable and controlling patient
dosing.
[0137] Proposed liquid formulations, e.g., for oral administration
of a number of drugs shown herein in the table have heretofore been
based primarily on the use of ethanol and oils or similar excipient
as carrier media. Thus, the commercially available drink-solutions
of a number of drugs employ ethanol and olive oil or corn oil as
carrier medium in conjunction with solvent systems comprising e.g.,
ethanol and LABRIFIL and equivalent excipient as carrier media. For
example, the commercially available Cyclosporin drink solution
employs ethanol and olive oil or corn oil as carrier medium in
conjunctions with a Labroid as a surfactant. See e.g., U.S. Pat.
No. 4,388,307. Use of the drink solution and similar composition as
proposed in the art is however accompanied by a variety of
difficulties.
[0138] Further, the palatability of the known oil based system has
proved problematic. The taste of the known drink-solution of
several drugs is, in particular, unpleasant. Admixture with an
appropriate flavored drink, for example, chocolate drink
preparation, at high dilution immediately prior to ingestion has
generally been practiced in order to make regular therapy at all
acceptable. Adoption of oil-based systems has also required the use
of high ethanol concentrations which is itself inherently
undesirable, in particular where administration to children is
foreseen. In addition, evaporation of the ethanol, e.g., from
capsules (adopted in large part, to meet problems of palatability,
as discussed or other forms (e.g., when opened)) results in the
development of a drug precipitate.
[0139] Where such compositions are presented in, for example, soft
gelatin encapsulated form, this particular difficulty necessitates
packaging of the encapsulated product in an air-tight component,
for example, an air-tight blister or aluminum-foil blister package.
This in turn renders the product both bulky and more expensive to
produce. The storage characteristics of the aforesaid formulations
are, in addition, far from ideal.
[0140] Bioavailability levels achieved using existing oral dosage
system for a number of drugs described herein are also low and
exhibit wide variation between individuals, individual patient
types and even for single individuals at different times during the
course of therapy. Reports in the literature indicate that
currently available therapy employing the commercially available
drug drink solution provides an average absolute bioavailability of
approximately 10-30% only, with a marked variation between
individual groups, e.g., between liver (relatively low
bioavailability) and bone-marrow (relatively high bioavailability)
transplant recipients. Reported variation in bioavailability
between subjects has varied from one or a few percent for some
patients, to as much as 90% or more for others. And as already
noted, marked change in bioavailability for individuals with time
is frequently observed. Thus, there is a need for a more uniform
and high bioavailability of a number drugs in patients.
[0141] Use of dosage forms of existing drugs is also characterized
by extreme variation in required patient dosing. To achieve
effective therapy, drug blood or blood serum levels have to be
maintained within a specified range. This required range can in
turn, vary, depending on the particular condition being treated,
e.g., whether therapy is to prevent one or more pharmacological
actions of a specific drug and when alternative therapy is employed
concomitantly with principal therapy. Because of the wide
variations in bioavailability levels achieved with conventional
dosage forms, daily dosages needed to achieve required blood serum
levels will also vary considerably from individual to individual
and even for a single individual. For this reason it may be
necessary to monitor blood/blood-serum levels of patients receiving
drug therapy at regular and frequent intervals. Monitoring of
blood/blood-serum levels has to be carried out on a regular basis.
This is inevitably time consuming and inconvenient and adds
substantially to the overall cost of therapy.
[0142] It is also the case that blood/blood serum levels of a
number of drugs without the amino acid linkage described herein
achieved using available dosage systems exhibit extreme variation
between peak and trough levels. That is, for each patient,
effective drug levels in the blood vary widely between
administrations of individual dosages.
[0143] There is also a need for providing a number of drugs
described herein, especially the beta-lactum antibiotics,
Cyclosporin, cephalosporins, steroids, quinolone antibiotics and
the like, in a water-soluble form for injection. It is well known
that Cremaphore L.RTM. (CreL) used in current formulations of a
number of drugs described hereinbelow is a polyoxyethylated
derivative of castor oil and is a toxic vehicle. There have been a
number of incidences of anaphylaxis due to the castor oil
component. At present there is no formulation that would allow many
of these drugs to be in aqueous solution at the concentrations
needed due to poor water solubility of the drug.
[0144] Beyond all these very evident practical difficulties lies
the occurrence of undesirable side reactions already alluded to,
observed employing available oral dosage forms.
[0145] Several proposals to meet these various problems have been
suggested in the art, including both solid and liquid oral dosage
forms. An overriding difficulty which has however remained is the
inherent insolubility of the several of the drugs without the amino
acid linkage shown in the table hereinbelow in aqueous media, hence
preventing the use of a dosage form which can contain the drugs in
sufficiently high concentration to permit convenient use and yet
meet the required criteria in terms of bioavailability, e.g.
enabling effective absorption from the stomach or gut lumen and
achievement of consistent and appropriately high blood/blood-serum
levels, is needed.
[0146] The particular difficulties encountered in relation to oral
dosing with these drugs have inevitably led to restrictions in the
use of specific drug therapy for the treatment of relatively less
severe or endangering disease conditions. For example, taking
Cyclosporin as a test drug, a particular area of difficulty in this
respect has been the adoption of Cyclosporin therapy in the
treatment of autoimmune diseases and other conditions affecting the
skin, for example for the treatment of atopic dermatitis and
psoriasis and, as also widely proposed in the art, for hair growth
stimulation, e.g. in the treatment of alopecia due to ageing or
disease.
[0147] Thus while oral Cyclosporin therapy has shown that the drug
is of considerable potential benefit to patients suffering e.g.
from psoriasis, the risk of side-reaction following oral therapy
has prevented common use. Various proposals have been made in the
art for application of Cyclosporins, e.g. Cyclosporin, in topical
form and a number of topical delivery systems have been described.
Attempts at topical application have however failed to provide any
demonstrably effective therapy.
[0148] However, the present invention overcomes the problems
described hereinabove. More specifically, the amino acid derivative
of the present invention significantly enhances its solubility in
aqueous solutions relative to the non-derivative form of the
pharmaceutical, thereby avoiding the need to utilize a carrier,
such as ethanol or castor oil when administered as a solution.
Moreover, the amino acid derivatives of these drugs, in accordance
with the present invention, do not exhibit the side effects of the
prior art formulations. Further, it has been found that when many
of the drugs in the table hereinbelow is administered in its amino
acid derivative form in accordance with the present invention,
there is enhanced oral absorption, thereby enhancing significantly
its bioavailability and its efficacy.
[0149] The preferred drugs used in combination with the amino acids
forming derivatives are listed hereinbelow in the following table
and the benefits found are as listed in the penultimate column of
the table. In the table, the key is as follows:
[0150] a) Improved taste smell
[0151] b) Desired Octanol/water partition coefficient (i.e.
solubility in water)
[0152] c) Improved stability in vitro and in vivo
[0153] d) Penetration of blood-brain barrier
[0154] e) Elimination of first pass effect in liver
[0155] f) Reduction of enterohepatic recirculation
[0156] g) Painless injections with parenteral formulations
[0157] h) Improved bioavailability
[0158] i) Increased rate of absorption
[0159] j) Reduced side effects
[0160] k) Dose proportionability
[0161] l) Selective hydrolysis of the derivative at site of
actions
[0162] m) Controlled release properties
[0163] n) Targeted drug delivery
[0164] o) Reduction in toxicity, hence improved therapeutic
ratio
[0165] p) Reduced dose
[0166] q) Alteration of metabolic pathway to deliver more drug at
site of action.
[0167] Moreover, the table indicates the utility of the derivative.
The utility of the derivative is the same as the corresponding drug
(without the amino acid moiety attached). The utility is described
in the literature such as in the Physicians Desk Reference, 2004
edition, the contents of which are incorporated by reference.
TABLE-US-00002 TABLE 1 Amino Acids that can Improvements be reacted
with the Most with Applicable Preferred Most drug to form the
Preferred derivatives Amino Acid Dose Dose Preferred
ster/amide/azo/anhydride Preferred Amino utility Derivatives of
Range Range Dose Range derivatives Amino Acids Acids immuno Utility
Cyclosporin All doses expressed as drug base Lys, Leu, Ile, Gly,
Lys, Leu, Ile, Gly, Lys, Pro, b, e, f, g, h, prophylaxis of organ
Preferred Asp, Glu, Met, Ala, Asp, Glu, Met, Ala, & Gly and k,
l, o, and p rejection, e.g., kidney, Forms Val, Pro, His, Tyr, Ser,
Val, Pro, His, Tyr, dipeptides liver and heart allogenic Oral
Tab/Cap 5-1000 mg 20-250 mg 25-100 mg Nor, Arg, Phe, Trp, Thr, Arg,
Phe, Trp, of Lys- transplants, treatment of Oral Liquid 1-25 mg/ml
5-15 mg/ml 10 mg/ml Hyp, Hsr, Car, Ort, Gln, Asn, Cys and Gly, Pro-
rheumatoid arthritis and IV Injections 10-250 mg 25-100 mg 50 mg/5
ml Cav, Asn, Gln, Can, Ser, Hyp, Sar or Gly, Gly- psoriosis per 5
ml per 5 ml Tau, Djk, GABA, Cys, dipeptides of Gly Dcy, Thr, and
Sar or combination of any dipeptide of two amino acids combination
of any especially AA-Gly, two amino acids where Gly is a especially
AA-Gly, spacer attached to where Gly is a spacer cyclosporin and
attached to AA is the above- cyclosporin and AA is cited amino
acids. the above-cited amino acids. Lopinavir All doses expressed
as drug base Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Lys, Pro, b,
h, j, k, treatment of HIV Preferred Asp, Glu, Met, Ala, Asp, Glu,
Met, Ala, Gly, & and o infections, e.g., AIDS Forms Val, Pro,
His, Tyr, Ser, Val, Pro, His, Tyr, Ala Oral Tab/Cap 0.1-1 gm
200-800 mg 400-500 mg Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, Oral
Liquid 0.1-1 gm/5 ml 0.2-0.8 g/ 400 mg/5 ml Hyp, Hsr, Car, Ort,
Gln, Asn, Cys and 5 ml Cav, Asn, Gln, Can, Ser, Hyp, Sar Tau, Djk,
GABA, Cys, Dcy, Thr, and Sar Cefdinir All doses expressed as drug
base Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Ser, a, b, e, f, h,
antibiotic treatment of diseases caused Preferred Asp, Glu, Met,
Ala, Asp, Glu, Met, Ala, Hyp, i, o, and p by Haemophilus
influenzae, including Forms Val, Pro, His, Tyr, Ser, Val, Pro, His,
Tyr, Tyr, & B-lactamase producing strains, e.g., Oral Tab/Cap
0.1-1 gm 0.2-0.5 gm 200-400 mg Nor, Arg, Phe, Trp, Thr, Arg, Phe,
Trp, Thr Haemophilus parainfluenzae (including Oral Liquid 0.1-1
gm/ 0.2-0.5 gm/ 0.2-0.4 gm/ Hyp, Hsr, Car, Ort, Gln, Asn, Cys and
.beta.-lactosamase producing strains) and 5 ml 5 ml 5 ml Cav, Asn,
Gln, Can, Ser, Hyp, Sar moraxella catarihalis (including .beta.- IV
Infusions 0.01-1 gm/ 20-500 mg/ 50-150 mg/ Tau, Djk, GABA, Cys,
lactamase producing strains), and 100 ml 100 ml 100 ml Dcy, Thr,
and Sar streptococcus pyogenes; such as pneumonia, bronchitis and
sinusitis, pharyngitis and tonsillitis Zileuton All doses expressed
as drug base Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Gly, Lys, b,
h, i, j, k, treatment of asthma Preferred Asp, Glu, Met, Ala, Asp,
Glu, Met, Ala, Sar, Ala, o, p Forms Val, Pro, His, Tyr, Ser, Val,
Pro, His, Tyr, Pro Oral Tab/Cap 200-1200 mg 200-800 mg 300-400 mg
Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, Oral Liquid 200-1200 mg/
200-800 mg/ 200-400 mg/ Hyp, Hsr, Car, Ort, Gln, Asn, Cys and 5 ml
5 ml 5 ml Cav, Asn, Gln, Can, Ser, Hyp, Sar Tau, Djk, GABA, Cys,
Dcy, Thr, and Sar Nelfinavir All Doses expressed as drug base Lys,
Leu, Ile, Gly, Lys, Leu, Ile, Gly, Gly, Lys, b, h, i, j, k,
treatment of HIV, infected patients, Preferred Asp, Glu, Met, Ala,
Asp, Glu, Met, Ala, Sar, Ala, o, p e.g., AIDS forms Val, Pro, His,
Tyr, Ser, Val, Pro, His, Tyr, Pro Oral Tab/Cap 0.05-1 gm 0.1-0.5 gm
0.2-0.4 gm Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, Oral Powder
10-250 mg/gm 20-200 mg/gm 40-100 mg/gm Hyp, Hsr, Car, Ort, Gln,
Asn, Cys and IV 10-250 mg/ 20-200 mg/ 40-100 mg/ Cav, Asn, Gln,
Can, Ser, Hyp, Sar Formulation 100 ml 100 ml 100 ml Tau, Djk, GABA,
Cys, Dcy, Thr, and Sar Flavoxate All doses expressed as drug base
Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Hyp, Ser, Tyr, b, h,
i, j, k, treatment of urinary spasms Preferred Forms Glu, Met, Ala,
Val, Pro, Asp, Glu, Met, Ala, & Thr l, o, & p Oral Tab/Cap
10-1000 mg 20-500 mg 50-250 mg His, Tyr, Ser, Nor, Arg, Val, Pro,
His, Tyr, Oral Liquid 10-1000 mg/ 20-500 mg/ 50-250 mg/ Phe, Trp,
Hyp, Hsr, Thr, Arg, Phe, Trp, 5 ml 5 ml 5 ml Car, Ort, Cav, Asn,
Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy,
Thr, and Sar Candesarten All doses expressed as drug base Lys, Leu,
Ile, Gly, Asp, Lys, Leu, Ile, Gly, Hyp, Ser, Tyr, b, c, e, f,
treatment of hypertension Preferred Forms Glu, Met, Ala, Val, Pro,
Asp, Glu, Met, Ala, & Thr h, i, j, k, l, Oral Tab/Cap 1-100 mg
2-75 mg 4-50 His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, o, p, q
Oral Liquid 1-100 mg/ 2-75 mg/ 4-50 mg/ Phe, Trp, Hyp, Hsr, Thr,
Arg, Phe, Trp, 5 ml 5 ml 5 ml Car, Ort, Cav, Asn, Gln, Asn, Cys and
Gln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and Sar
Propofol All doses expressed as drug base Lys, Leu, Ile, Gly, Asp,
Lys, Leu, Ile, Gly, Gly, Lys, Sar, b, c, d, g, provides central
nervous Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met,
Ala, Pro, Ala, & h, j, k, l, system anesthesia IV Infusions
1-25 mg/ml 2.0-20 mg/ml 5-15 mg/ml His, Tyr, Ser, Nor, Arg, Val,
Pro, His, Tyr, Val m, n, o, p, q Phe, Trp, Hyp, Hsr, Thr, Arg, Phe,
Trp, Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser,
Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Nisoldipine All doses
expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,
Gly, Lys, Ser, b, e, h, i, j, o calcium channel blocker, Preferred
Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, & Hyp
treatment of hypertension Oral Tab/Cap 2-100 mg 2.5-75 mg 5-50 mg
His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid 2-100 mg/
2.5-75 mg/ 5-50 mg/ Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, 5 ml 5
ml 5 ml Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk,
Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Amlodipine All Doses
expressed as drug base Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Gly,
Lys, Ser, b, e, h, i, j, o calcium channel blocker, Preferred forms
IV Asp, Glu, Met, Ala, Asp, Glu, Met, Ala, & Hyp treatment of
hypertension Oral Tab/Cap 0.1-20 mg 1-10 mg 2.5-5 mg Val, Pro, His,
Tyr, Ser, Val, Pro, His, Tyr, Oral Liquid 0.1-20 mg/ 1-10 mg/ 2.5-5
mg/ Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, 5 ml 5 ml 5 ml Hyp,
Hsr, Car, Ort, Gln, Asn, Cys and Cav, Asn, Gln, Can, Ser, Hyp, Sar
Tau, Djk, GABA, Cys, Dcy, Thr, and Sar Ciprofloxacin All doses
expressed as drug base Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Hyp,
Ser, a, b, c, g, Antibiotic; inhibits various Preferred Forms Asp,
Glu, Met, Ala, Asp, Glu, Met, Ala, Thr, Gly, & h, i, j, k, o, p
bacteria, e.g., pseudomonas Oral Tab/Cap 0.1-1.5 gm 0.1-1.0 gm
0.2-0.8 gm Val, Pro, His, Tyr, Ser, Val, Pro, His, Tyr, Lys
aeruginosa, staphylococcus Oral Liquid 0.05-1 gm/ 0.08-1 gm/ 0.12-1
g/5 ml Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, aureus or proteus
mirabilis; 5 ml 5 ml Hyp, Hsr, Car, Ort, Gln, Asn, Cys and
treatment of corneal ulcers, IV Bulk (Sterile) 2-25 mg/ml 3-20
mg/ml 5-15 mg/ml Cav, Asn, Gln, Can, Ser, Hyp, Sar conjunctivitis,
acute otitis Tau, Djk, GABA, Cys, externa, Dcy, Thr, and Sar
Ramipril All doses expressed as drug base Lys, Leu, Ile, Gly, Lys,
Leu, Ile, Gly, Hyp, Ser, j, o treatment of hypertension Preferred
Forms Asp, Glu, Met, Ala, Asp, Glu, Met, Ala, Thr, Gly, & Oral
Tab/Cap 0.1-20 mg 0.5-12 mg 1-10 mg Val, Pro, His, Tyr, Ser, Val,
Pro, His, Tyr, Lys Oral Liquid 0.1-20 mg/ 0.5-12 mg/ 1-10 mg/5 ml
Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, 5 ml 5 ml Hyp, Hsr, Car,
Ort, Gln, Asn, Cys and Cav, Asn, Gln, Can, Ser, Hyp, Sar Tau, Djk,
GABA, Cys, Dcy, Thr, and Sar Trandolapril All doses expressed as
drug base Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Ser, Hyp, j, o
treatment of hypertension Preferred Forms Asp, Glu, Met, Ala, Asp,
Glu, Met, Ala, Thr, Gly & Oral Tab/Cap 0.1-10 mg 0.5-7.5 mg 1-4
mg Val, Pro, His, Tyr, Ser, Val, Pro, His, Tyr, Lys Oral Liquid
0.1-10 mg/ 0.5-7.5 mg/ 1-4 mg/5 ml Nor, Arg, Phe, Trp, Thr, Arg,
Phe, Trp, 5 ml 5 ml Hyp, Hsr, Car, Ort, Gln, Asn, Cys and Cav, Asn,
Gln, Can, Ser, Hyp, Sar Tau, Djk, GABA, Cys, Dcy, Thr, and Sar
Fosinopril All doses expressed as drug base Lys, Leu, Ile, Gly,
Lys, Leu, Ile, Gly, Ser, Hyp, j, o treatment of hypertension
Preferred Forms Asp, Glu, Met, Ala, Asp, Glu, Met, Ala, Thr, Gly,
& Oral Tab/Cap 1-100 mg 2-75 mg 5-50 mg Val, Pro, His, Tyr,
Ser, Val, Pro, His, Tyr, Lys Oral Liquid 1-100 mg/ 2-75 mg/5 ml
5-50 mg/ Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, 5 ml 5 ml Hyp,
Hsr, Car, Ort, Gln, Asn, Cys and Cav, Asn, Gln, Can, Ser, Hyp, Sar
Tau, Djk, GABA, Cys, Dcy, Thr, and Sar Enalapril All Doses
expressed as drug base Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Ser,
Hyp, j, o treatment of hypertension Preferred forms Asp, Glu, Met,
Ala, Asp, Glu, Met, Ala, Thr, Gly & Oral Tab/Cap 0.5-100 mg
1-50 mg 2-25 mg Val, Pro, His, Tyr, Ser, Val, Pro, His, Tyr, Lys
Oral Liquid 0.5-100 mg/ 1-50 mg/5 ml 2-25 mg/ Nor, Arg, Phe, Trp,
Thr, Arg, Phe, Trp, 5 ml 5 ml Hyp, Hsr, Car, Ort, Gln, Asn, Cys and
Cav, Asn, Gln, Can, Ser, Hyp, Sar Tau, Djk, GABA, Cys, Dcy, Thr,
and Sar Benazepril All doses expressed as drug base Lys, Leu, Ile,
Gly, Lys, Leu, Ile, Gly, Hyp, Ser, j, o treatment of hypertension
Preferred Forms Asp, Glu, Met, Ala, Asp, Glu, Met, Ala, Thr, Gly,
& Oral Tab/Cap 1-100 mg 2-75 mg 2.5-50 mg Val, Pro, His, Tyr,
Ser, Val, Pro, His, Tyr, Lys Oral Liquid 1-100 mg/ 2-75 mg/5 ml
2.5-50 mg/ Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, 5 ml 5 ml Hyp,
Hsr, Car, Ort, Gln, Asn, Cys and Cav, Asn, Gln, Can, Ser, Hyp, Sar
Tau, Djk, GABA, Cys, Dcy, Thr, and Sar Perindopril All doses
expressed as drug base Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Hyp,
Ser, j, o treatment of hypertension Preferred Forms Asp, Glu, Met,
Ala, Asp, Glu, Met, Ala, Thr, Gly, & Oral Tab/Cap 0.1-20 mg
0.5-15 mg 1-10 mg Val, Pro, His, Tyr, Ser, Val, Pro, His, Tyr, Lys
Oral Liquid 0.1-20 mg/ 0.5-15 mg/5 ml 1-10 mg/ Nor, Arg, Phe, Trp,
Thr, Arg, Phe, Trp, 5 ml 5 ml Hyp, Hsr, Car, Ort, Gln, Asn, Cys and
Cav, Asn, Gln, Can, Ser, Hyp, Sar Tau, Djk, GABA, Cys, Dcy, Thr,
and Sar Moexipril All doses expressed as drug base Lys, Leu, Ile,
Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, j, o treatment of
hypertension Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu,
Met, Ala, Gly & Lys Oral Tab/Cap 1-30 mg 2-20 mg 5-15 mg His,
Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid 1-30 mg/5 ml
2-20 mg/5 ml 5-15 mg/ Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, 5 ml
Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser, Hyp,
Sar GABA, Cys, Dcy, Thr, and Sar Cromolyn All doses expressed as
drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp,
Thr, & b, c, h, i, j, inhibits release of histamine Preferred
Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Pro k, l, n, o,
and leukotrienes from mast Oral Tab/Cap 10-200 mg 20-100 mg 20-50
mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, p, q cell;
treatment of Oral Liquid 10-200 mg/ 20-100 mg/ 20-50 mg/ Phe, Trp,
Hyp, Hsr, Thr, Arg,
Phe, Trp, mastocytosis, asthma 5 ml 5 ml 5 ml Car, Ort, Cav, Asn,
Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy,
Thr, and Sar Amoxicillin All Doses expressed as drug base Lys, Leu,
Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, a, b, c, h,
antibiotic effective against Preferred forms Glu, Met, Ala, Val,
Pro, Asp, Glu, Met, Ala, Gly & Lys i, j, k, l, o, p
.beta.-lactamase negative strains Oral Tab/Cap* 0.1-1.5 gm 0.2-1.2
gm 0.25-1 gm His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, causing
infections of ear, Oral Liquid 0.1-1.5 gm/ 0.2-1.2 gm/ 0.25-1 gm/
Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, nose, throat, e.g., 5 ml 5
ml 5 ml Car, Ort, Cav, Asn, Gln, Asn, Cys and streptococcus, Oral
Powder 0.1-0.75 gm 0.1-0.6 gm 0.125-0.5 gm Gln, Can, Tau, Djk, Ser,
Hyp, Sar staphylococcus or H (*also chewable) GABA, Cys, Dcy, Thr,
influenzae; treatment of and Sar infections of genitourinary tract
due to E. coli, P. mirabilis, E. faecalis, infections of skin due
to streptococcus, staphylococcus or E. coli, infections of lower
respiratory tract due to streptoccus, staphyloccus or H.
influenzae, and gonorrhea Cefuroxime All doses expressed as drug
base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Hyp, Ser, Thr, b,
c, e, f, antibiotic; treatment of Preferred Forms Glu, Met, Ala,
Val, Pro, Asp, Glu, Met, Ala, Gly, & Lys h, i, j, k, o, p
pharyngitis/tonsillitis caused Oral Tab/Cap 10-1000 mg 50-750 mg
100-600 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, by
streptococcus, acute Oral Liquid 10-1000 mg/ 50-750 mg/ 100-600 mg/
Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, bacterialotitis media
caused 5 ml 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and by
streptococcus, H Tau, Djk, GABA, Cys, Ser, Hyp, Sar influenzae,
moraxella Dcy, Thr, and Sar catarihalis or streptococcus, urinary
tract infections caused by E. coli or Klebsiella pneumonia,
gonorrhea, skin infections cause by staphylococcus or straptococcus
Ceftazidime All doses expressed as drug base Lys, Leu, Ile, Gly,
Asp, Lys, Leu, Ile, Gly, Hyp, Ser, Thr, a, b, c, g, antibiotic,
treatmtent of Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu,
Met, Ala, Gly, & Lys h, i, j, k, l, lower respiratory tract
Powder for IV 0.1-5 gm 0.25-4 gm 0.5-2 gm His, Tyr, Ser, Nor, Arg,
Val, Pro, His, Tyr, o, p, q infections, including Oral Tab/Cap
0.1-1 gm 0.25-1 gm 0.5-1 gm Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe,
Trp, pneumonia caused by Oral Liquid 0.1-2.5 gm/ 0.25-2 gm/ 0.5-1
gm/ Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and pseudomonas, H.
influenzae, 5 ml 5 ml 5 ml Tau, Djk, GABA, Cys, Ser, Hyp, Sar
Klebsiella, Dcy, Thr, and Sar Enterbacter, E. coli, proteus
mirabilis, streptococcus, staphylococcus; skin and skin structure
infections, caused by pseudomonas aeruginosa, Klebsiella, E. coli,
Proteus enterbacter, staphylococcus, streptococcus, urinary tract
infections casued by pseudomonas aeruginosa, enterbacter, proteus,
Klebsiella, E. coli; bone and joint infections caused by
pseudomonas, eruginosa, Klebsiella, Enterbacter, or staphylococcus;
gynecologic infections including endometritis, pelvic cellulits and
infections of the female genital tract caused by E. coli,
intra-abdominal infections and central nervous system infections,
including meningitis Cefpodoxime All doses expressed as drug base
Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, a, b,
c, g, h, i, antibiotic especially Preferred Forms Glu, Met, Ala,
Val, Pro, Asp, Glu, Met, Ala, Gly & Lys j, k, l, o, p, q
against streptococcus, Oral Tab/Cap 10-500 mg 25-350 mg 50-250 mg
His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, H. influenzae, Oral
Liquid 10-500 mg/ 25-350 mg/ 50-250 mg/ Phe, Trp, Hyp, Hsr, Car,
Thr, Arg, Phe, Trp, moraxella catarrhalis; 5 ml 5 ml 5 ml Ort, Cav,
Asn, Gln, Can, Gln, Asn, Cys and treatment of acute otis Tau, Djk,
GABA, Cys, Ser, Hyp, Sar media, pharyngitis, Dcy, Thr, and Sar
tonsillitis, pneumonia, bronchitis, gonorrhea, and rectal
infections in women Atovaquone All doses expressed as drug base
Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Sar, a, b,
h, i, j, k, treatment of malaria Preferred Forms Glu, Met, Ala,
Val, Pro, Asp, Glu, Met, Ala, Ala, Pro & Ser o, p caused by
plasmodium Oral Tab/Cap 50-1000 mg 100-500 mg 200-300 mg His, Tyr,
Ser, Nor, Arg, Val, Pro, His, Tyr, parasite Oral Liquid above/5 ml
above/5 ml above/5 ml Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp,
For Pediatric Use 10-150 mg/ 25-100 mg/ 50-75 mg/ Ort, Cav, Asn,
Gln, Can, Gln, Asn, Cys and 5 ml 5 ml 5 ml Tau, Djk, GABA, Cys,
Ser, Hyp, Sar Dcy, Thr, and Sar Acyclovir All Doses expressed as
drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Sar,
Hyp b, c, h, i, j, k, treatment of human Preferred forms Glu, Met,
Ala, Val, Pro, Asp, Glu, Met, Ala, Pro & Ser o, p
cytomegalovirus Oral Tab/Cap 50-1000 mg 100-750 ml 150-500 mg His,
Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, (HCMV) Oral Liquid 50-1000
mg/ 100-750 mg/ 150-500 mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe,
Trp, 5 ml 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau,
Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Gancyclovir All
doses expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu,
Ile, Gly, Hyp, Ser, Thr, b, c, e, f, h, i, j, treatment of human
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly,
& Lys k, o, p cytomegalo virus Oral Tab/Cap 0.1-1 gm 0.2-0.8 gm
0.2-0.6 gm His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, (HCMV) Oral
Liquid 0.1-1 gm/5 ml 0.2-0.8 mg/ 0.2-0.6 mg/ Phe, Trp, Hyp, Hsr,
Car, Thr, Arg, Phe, Trp, 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln,
Asn, Cys and IV Infusions 10-200 mg/ml 25-100 mg/ml 30-60 mg/ml
Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Penciclovir
All doses expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu,
Ile, Gly, Hyp, Ser, Thr, b, c, e, f, h, i, j, treatment of human
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly,
& Lys k, o, p cytomegalovirus Powder for IV 10-1000 mg/ml
25-750 mg/ml 50-500 mg/ml His, Tyr, Ser, Nor, Arg, Val, Pro, His,
Tyr, (HCMV) Topical Cream 0.1-5% 0.25-3% 0.5-2.5% Phe, Trp, Hyp,
Hsr, Car, Thr, Arg, Phe, Trp, Oral Cap/Tab 10-500 mg 20-300 mg
25-250 mg Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk,
GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Niacin ER All doses
expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,
Ser, Hyp, Thr, a, b, h, i, j, l, lipid management Preferred Forms
Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Tyr, Gly & Lys m,
n, o, p, q Oral Tab/Cap 0.2-2 gm 0.25-1.5 gm 0.5-1 gm His, Tyr,
Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid 0.2-2 gm/5 ml 0.25-1
gm/ 0.5-1 gm/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, 5 ml 5
ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys,
Ser, Hyp, Sar Dcy, Thr, and Sar Bexarotene All doses expressed as
drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp,
Thr, b, c, h, i, j, k, l, treatment of skin Preferred Forms Glu,
Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly, & Lys o, p
conditions, especially Oral Tab/Cap 10-500 mg 25-250 mg 50-100 mg
His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, those requiring Oral
Liquid above/5 ml above/5 m above/5 ml Phe, Trp, Hyp, Hsr, Car,
Thr, Arg, Phe, Trp, activation of retinoid Topical Gel 0.1-5%
0.25-2.5% l 0.5-1.5% Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and X
receptors Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar
Propoxyphene All Doses expressed as drug base Lys, Leu, Ile, Gly,
Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, a, b, c, h, i, j, treatment
of pain Preferred forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met,
Ala, Gly, & Lys k, l, o, p Oral Tab/Cap 20-400 mg 25-250 mg
30-150 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid
20-400 mg/ 25-250 mg/ 30-150 mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg,
Phe, Trp, 5 ml 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and
Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Salsalate All
doses expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu,
Ile, Gly, Hyp, Ser, Thr, Gly, b, c, h, i, j, treatment of Preferred
Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, & Lys k, o,
p inflammatory Oral Tab/Cap 0.2-2 gm 0.25-1.5 gm 0.3-1 gm His, Tyr,
Ser, Nor, Arg, Val, Pro, His, Tyr, conditions Oral Liquid 0.2-2
gm/5 ml 0.25-1.5 gm/ 0.3-1 gm/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg,
Phe, Trp, 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau,
Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Acetaminophen All
doses expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu,
Ile, Gly, Hyp, Ser, Sar, Gly, a, b, c, e, h, treatment of pain or
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, &
Lys i, j, k, o, p fever Oral Tab/Cap 20-1000 mg 50-800 mg 100-600
mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid 20-1000
mg/ 50-800 mg/ 100-600 mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe,
Trp, 5 ml 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau,
Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Ibuprofen All doses
expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,
Ser, Hyp, Thr, Tyr, a, b, h, i, j, l, treatment of pain, fever
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly
& Lys m, n, o, p, q or inflammation Oral Tab/Cap 20-1000 mg
50-800 mg 100-600 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr,
Oral Liquid 20-1000 mg/ 50-800 mg/ 100-600 mg/ Phe, Trp, Hyp, Hsr,
Car, Thr, Arg, Phe, Trp, 5 ml 5 ml 5 ml Ort, Cav, Asn, Gln, Can,
Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and
Sar Lovastatin All doses expressed as drug base Lys, Leu, Ile, Gly,
Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Gly, b, c, e, f, lowers
choloesterol Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu,
Met, Ala, & Lys h, i, j, k, concentration; Oral Tab/Cap 1-100
mg 2-80 mg 5-50 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, l,
o, p inhibits HMG-CoA Oral Liquid 1-100 mg/5 ml 2-80 mg/ 5-50 mg/
Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, reductase 5 ml 5 ml Car,
Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser, Hyp, Sar
GABA, Cys, Dcy, Thr, and Sar Simavastatin All Doses expressed as
drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp,
Thr, Gly, b, c, e, f, lowers choloesterol Preferred forms Glu, Met,
Ala, Val, Pro, Asp, Glu, Met, Ala, & Lys h, i, j, k,
concentration; Oral Tab/Cap 1-200 mg 2-150 mg 2.5-100 mg His, Tyr,
Ser, Nor, Arg, Val, Pro, His, Tyr, l, o, p inhibits HMG-CoA Oral
Liquid 1-200 mg/5 ml 2-150 mg/ 2.5-100 mg/ Phe, Trp, Hyp, Hsr, Thr,
Arg, Phe, Trp, reductase 5 ml 5 ml Car, Ort, Cav, Asn, Gln, Asn,
Cys and Gln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and
Sar Atorvastatin All doses expressed as drug base Lys, Leu, Ile,
Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Gly, b, c, e, f,
lowers choloesterol Preferred Forms Glu, Met, Ala, Val, Pro, Asp,
Glu, Met, Ala, & Lys h, i, j, k, concentration; Oral Tab/Cap
1-250 mg 2-125 mg 5-100 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His,
Tyr, l, o, p inhibits HMG-CoA Oral Liquid 1-250 mg/5 ml 2-125 mg/
5-100 mg/ Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, reductase 5 ml 5
ml Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser,
Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Pravastatin All doses
expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys,
Leu, Ile, Gly, Ser, Hyp, Thr, Gly, b, c, e, f, lowers choloesterol
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, &
Lys h, i, j, k, concentration; Oral Tab/Cap 1-250 mg 2-125 mg 5-75
mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, l, o, p inhibits
HMG-CoA Oral Liquid 1-250 mg/5 ml 2-125 mg/ 5-75 mg/ Phe, Trp, Hyp,
Hsr, Thr, Arg, Phe, Trp, reductase 5 ml 5 ml Car, Ort, Cav, Asn,
Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy,
Thr, and Sar Fluvastatin All doses expressed as drug base Lys, Leu,
Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Tyr, b, c, e, f,
lowers choloesterol Preferred Forms Glu, Met, Ala, Val, Pro, Asp,
Glu, Met, Ala, Gly & Lys h, i, j, k, concentration; Oral
Tab/Cap 1-250 mg 2-125 mg 5-75 mg His, Tyr, Ser, Nor, Arg, Val,
Pro, His, Tyr, l, o, p inhibits HMG-CoA Oral Liquid 1-250 mg/5 ml
2-125 mg/ 5-75 mg/ Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp,
reductase 5 ml 5 ml Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can,
Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Nadolol All
doses expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu,
Ile, Gly, Lys, Gly, Sar, Ser, b, h, i, j, treatment of angina
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, &
Pro k, l, o, p pectoris and Oral Tab/Cap 1-250 mg 5-225 mg 10-200
mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, hypertension;
.beta.- Oral Liquid 1-250 mg/5 ml 5-225 mg/ 10-200 mg/ Phe, Trp,
Hyp, Hsr, Thr, Arg, Phe, Trp, adrenergic receptor 5 ml 5 ml Car,
Ort, Cav, Asn, Gln, Asn, Cys and antagonist Gln, Can, Tau, Djk,
Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Valsartan All Doses
expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,
Hyp, Ser, Thr, Lys, b, f, i, j, treating hypertension, Preferred
forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly & Sar k,
l, o, p angiotension II Oral Tab/Cap 10-500 mg 25-250 mg 50-200 mg
His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, antagonist Oral Liquid
10-500 mg/ 25-250 mg/ 50-200 mg/ Phe, Trp, Hyp, Hsr, Thr, Arg, Phe,
Trp, 5 ml 5 ml 5 ml Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can,
Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and Sar
Methylphenidate All doses expressed as drug base Lys, Leu, Ile,
Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Hyp, Sar, a, b, c, h,
treatment of attention Preferred Forms Glu, Met, Ala, Val, Pro,
Asp, Glu, Met, Ala, & Ser j, k, l, o, p deficit disorders and
Oral Tab/Cap 1-50 mg 2-40 mg 2.5-25 mg His, Tyr, Ser, Nor, Arg,
Val, Pro, His, Tyr, narcolepsy Oral Liquid 1-50 mg/5 ml 2-40 mg/
2.5-25 mg/5 ml Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, 5 ml Car,
Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser, Hyp, Sar
GABA, Cys, Dcy, Thr, and Sar Trovafloxacin All doses expressed as
drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly,
Ser, Pro, a, b, e, h, j, antibiotic; inhibits Preferred Forms Glu,
Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Hyp & Thr k, o, p
bacteria such as E. coli, Oral Tab/Cap 10-500 mg 50-300 mg 80-250
mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, pseudomonas, Oral
Liquid 10-500 mg/ 50-300 mg/ 80-250 mg/ Phe, Trp, Hyp, Hsr, Car,
Thr, Arg, Phe, Trp, aeruginosa, H. influenzae, 5 ml 5 ml 5 ml Ort,
Cav, Asn, Gln, Can, Gln, Asn, Cys and streptococous, Tau, Djk,
GABA, Cys, Ser, Hyp, Sar Klebsiella, Dcy, Thr, and Sar
staphylococcus, mycoplasma pneumoniae, peptostreptococcus,
prevotella; treatment of pneumonia, postsurgical infections;
gynecolgic and pelvic infections, such as endomyometritis,
parametritis, septic abortions, and post- partum infections; skin
infections, e.g., diabetic foot infections 5-AS* All doses
expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,
Glu, Gly, Tyr & Lys b, c, i, j, l, treatment of Preferred Forms
Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, m, n, o, p, q
tuberculosis Oral Tab/Cap 1-200 mg 5-150 mg 10-125 mg His, Tyr,
Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid 1-200 mg/5 ml 5-150
mg/ 10-125 mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, (*
5-Amino- 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and
Salicylic acid) Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and
Sar Methylprednisolone All doses expressed as drug base Lys, Leu,
Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Pro, b, c, g, j, l,
treatment of Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu,
Met, Ala, Sar & Ser m, n, o, p, q inflammation IM Injection
2-200 mg/ml 5-150 mg/ml 10-100 mg/ml His, Tyr, Ser, Nor, Arg, Val,
Pro, His, Tyr, especially from Topical Cream 0.001-5% 0.01-2.5%
0.1-2% Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, infections,
tissue Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and damage, allergy
and Tau, Djk, GABA, Cys, Ser, Hyp, Sar auto-immune Dcy, Thr, and
Sar disease MedroxyProgesterone All Doses expressed as drug base
Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Pro, b, c,
g, j, l, providing Preferred forms Glu, Met, Ala, Val, Pro, Asp,
Glu, Met, Ala, Sar & Ser m, n, o, p, q contraception IM
Injections 1 mg-4 gm/ml 10 mg-2 gm/ml 40 mg-1 gm/ml His, Tyr, Ser,
Nor, Arg, Val, Pro, His, Tyr, Phe, Trp, Hyp, Hsr, Car, Thr, Arg,
Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk,
GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Estramustine All doses
expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,
Gly, Lys, Pro, Ala, b, c, h, i, j, treatment of cancer Preferred
Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Sar & Val k,
l, o, p especially treatment Oral Tab/Cap 10-500 mg 25-250 mg
50-200 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, of
metastatic or Oral Liquid 10-500 mg/ 25-250 mg/ 50-200 mg/ Phe,
Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, progressive 5 ml 5 ml 5 ml
Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and carcinoma of Tau, Djk,
GABA, Cys, Ser, Hyp, Sar prostate Dcy, Thr, and Sar Miglitol All
doses expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu,
Ile, Gly, Lys, Gly, Sar, Pro, & b, c, i, j, n, q treatment of
type II Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met,
Ala, Ser diabetes Oral Tab/Cap 1-250 mg 2-150 mg 10-125 mg His,
Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid 1-250 mg/5 ml
2-150 mg/ 10-125 mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, 5
ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA,
Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Mefloquine All doses expressed
as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly,
Sar, Pro, a, b, c, h, i, treatment of malaria Preferred Forms Glu,
Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Val, Ala j, k, l, o, p, q
Oral Tab/Cap 10-500 mg 100-400 mg 150-300 mg His, Tyr, Ser, Nor,
Arg, Val, Pro, His, Tyr, Oral Liquid 10-500 mg/ 25-400 mg/ 150-300
mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, 5 ml 5 ml 5 ml
Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys,
Ser, Hyp, Sar Dcy, Thr, and Sar Danazol All Doses expressed as drug
base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Hyp, Pro, Ala,
Val, a, b, c, e, f, treatment of Preferred forms Glu, Met, Ala,
Val, Pro, Asp, Glu, Met, Ala, Ser & Thr g, h, i, j, k,
endometriosis and Oral Tab/Cap 2-500 mg 10-350 mg 25-250 mg His,
Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, l, n, o, p, q fibrostatic
breast Oral Liquid 2-500 mg/ 10-350 mg/ 25-250 mg/ Phe, Trp, Hyp,
Hsr, Car, Thr, Arg, Phe, Trp, disease 5 ml 5 ml 5 ml Ort, Cav, Asn,
Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy,
Thr, and Sar Eprosartan All doses expressed as drug base Lys, Leu,
Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Lys, b, c, h, i,
j, ACE inhibitors, Preferred Forms Glu, Met, Ala, Val, Pro, Asp,
Glu, Met, Ala, Gly & Val k, l, o, p treatment of Oral Tab/Cap
0.1-1 gm 200-800 mg 300-750 mg His, Tyr, Ser, Nor, Arg, Val, Pro,
His, Tyr, hypertension Oral Liquid 0.1-1 gm/ 200-800 mg/ 300-750
mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, 5 ml 5 ml 5 ml
Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys,
Ser, Hyp, Sar Dcy, Thr, and Sar Divalproex Na All doses expressed
as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp,
Thr, Lys, a, b, c, f, h, treatment of epilepsy Preferred Forms Glu,
Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly, & Val i, j, k, l,
o, p Oral Tab/Cap 50-800 mg 75-750 mg 100-600 mg His, Tyr, Ser,
Nor, Arg, Val, Pro, His, Tyr, Oral Liquid 50-800 mg/ 75-750 mg/
100-60 mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, 5 ml 5 ml 5
ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys,
Ser, Hyp, Sar Dcy, Thr, and Sar Fenofibrate All doses expressed as
drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp,
Thr, Lys, b, c, h, i, j, treatment of Preferred Forms Glu, Met,
Ala, Val, Pro, Asp, Glu, Met, Ala, Gly, & Ala k, l, o, p, q
hypercholestemia Oral Tab/Cap 10-800 mg 20-750 mg 100-600 mg His,
Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid 10-800 mg/
20-750 mg/ 100-600 mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp,
5 ml 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk,
GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Gabapentin All Doses
expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,
Cyclic Deriv. & Tyr b, c, d, e, f, treatment of Preferred Forms
Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, h, i, j, k, l,
convulsions Oral Tab/Cap 10-800 mg 25-750 mg 50-500 mg His, Tyr,
Ser, Nor, Arg, Val, Pro, His, Tyr, n, o, p, p Oral Liquid 10-800
mg/ 25-750 mg/ 50-500 mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe,
Trp, 5 ml 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau,
Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Lansoprazole All
doses expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu,
Ile, Gly, Gly, Lys, Pro, Sar, b, e, f, h, i, suppression of gastric
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Ser
& Val j, k, l, o, p acid secretion by Oral Tab/Cap 1-60 mg 2-50
mg 10-40 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Car,
inhibition of (H+, K+) Oral Liquid 1-6-mg/ 2-50 mg/5 ml 10-40 mg/
Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, ATP-ase enzyme 5 ml 5 ml
Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and system at the secretory
Tau, Djk, GABA, Cys, Ser, Hyp, Sar surface of the gastric Dcy, Thr,
and Sar parietal cell; treatment of gastric hyperacidity Omeprazole
All doses expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu,
Ile, Gly, Lys, Gly, Val, Pro & b, e, f, h, i, suppression of
gastric Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met,
Ala, Sar j, k, l, o, p acid secretion by Oral Tab/Cap 1-200 mg
2-100 mg 5-60 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr,
inhibition of(H+, K+) Oral Liquid 1-200 mg/ 2-100 mg/ 5-60 mg/ Phe,
Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, ATP-ase enzyme 5 ml 5 ml 5
ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and system at the
secretory Tau, Djk, GABA, Cys, Ser, Hyp, Sar surface of the gastric
Dcy, Thr, and Sar parietal cell, treatment of gastric hyperacidity
Megestrol All doses expressed as drug base Lys, Leu, Ile, Gly, Asp,
Lys, Leu, Ile, Gly, Gly, Lys, Sar, Pro, b, c, h, i, j, treatment of
anorexia; Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met,
Ala, Ser & Ala k, l, n, o, p improving appetite in Oral Tab/Cap
2-100 mg 4-80 mg 20-60 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His,
Tyr, anorexic and patients Oral Liquid 2-100 mg/ 4-80 mg/5 ml 20-60
mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, suffering from
AIDS 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk,
GABA, Cys, Ser, Hyp, Sar
Dcy, Thr, and Sar Metformin All doses expressed as drug base Lys,
Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Asp, Glu, Lys & Azo o,
p treatment of Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu,
Met, Ala, dimer hyperglycemia; aids Oral Tab/Cap 0.2-3 gm 0.25-1.5
gm 0.5-1 gm His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, insulin to
improve Oral Liquid 0.2-1 gm/ 0.25-1.5 mg/ 0.5-1 gm/ Phe, Trp, Hyp,
Hsr, Car, Thr, Arg, Phe, Trp, transport of glucose 5 ml 5 ml 5 ml
Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and into cells Tau, Djk,
GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Tazarotene All doses
expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,
Ser, Hyp, Thr, Lys, & B, c, h, I, j, treatment of psoriasis
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly k,
l, o, p and acne especially Topical Gel 0.01-0.3% 0.02-0.25%
0.025-0.125% His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, those
caused by Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, pathogenic
Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and microorganisms, allergy
Tau, Djk, GABA, Cys, Ser, Hyp, Sar and inflammation Dcy, Thr, and
Sar Sumatriptan All Doses expressed as drug base Lys, Leu, Ile,
Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Ala, Pro, b, c, d, g, h, 5
HT subtype receptor Preferred Forms Glu, Met, Ala, Val, Pro, Asp,
Glu, Met, Ala, Sar & Val i, j, k, l, n, agonist, treatment of
Oral Tab/Cap 5-250 mg 10-200 mg 20-125 mg His, Tyr, Ser, Nor, Arg,
Val, Pro, His, Tyr, o, p, q migraine headaches Oral Liquid 5-250
mg/ 10-200 mg/ 20-125 mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe,
Trp, 5 ml 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and IM
Injections 1-36 mg/ml 2-24 mg/ml 4-20 mg/ml Tau, Djk, GABA, Cys,
Ser, Hyp, Sar Dcy, Thr, and Sar Naratriptan All doses expressed as
drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly,
Sar, Val, b, h, i, j, k, 5 HT subtype Preferred Forms Glu, Met,
Ala, Val, Pro, Asp, Glu, Met, Ala, Ala, & Pro l, o, p receptor
agonist; Oral Tab/Cap 0.1-10 mg 0.25-5 mg 0.5-4 mg His, Tyr, Ser,
Nor, Arg, Val, Pro, His, Tyr, treatment of Oral Liquid 0.1-10 mg/
0.25-5 mg/ 0.5-4 mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp,
migraine headaches 5 ml 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln,
Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar
Zolmitriptan All doses expressed as drug base Lys, Leu, Ile, Gly,
Asp, Lys, Leu, Ile, Gly, Lys, Gly, Sar, Val, b, h, i, j, k, 5 HT
subtype Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met,
Ala, Ala, & Pro l, o, p receptor Oral Tab/Cap 0.1-12 mg 0.5-10
mg 1-7.5 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, agonist;
treatment of Oral Liquid 1-12 mg/ 0.5-10 mg/ 1-7.5 mg/ Phe, Trp,
Hyp, Hsr, Car, Thr, Arg, Phe, Trp, migraine headaches 5 ml 5 ml 5
ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys,
Ser, Hyp, Sar Dcy, Thr, and Sar Aspirin All doses expressed as drug
base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr,
Lys, a, b, c, e, f, antipyretic, anti- Preferred Forms Glu, Met,
Ala, Val, Pro, Asp, Glu, Met, Ala, Gly, & Ala g, h, j, k, l,
inflammatory, Oral Tab/Cap 10-1000 mg 20-800 mg 25-600 mg His, Tyr,
Ser, Nor, Arg, Val, Pro, His, Tyr, m, n, o, p, q analgesic, Oral
Liquid 10-1000 mg/ml 20-800 mg/ml 25-600 mg/ml Phe, Trp, Hyp, Hsr,
Car, Thr, Arg, Phe, Trp, thrombolytic; Ort, Cav, Asn, Gln, Can,
Gln, Asn, Cys and treatment of Tau, Djk, GABA, Cys, Ser, Hyp, Sar
hyperthermia, Dcy, Thr, and Sar myocardial infarction and
thrombolysis Olmesartan All doses expressed as drug base Lys, Leu,
Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Lys, b, h, i, j,
k, l, ACE inhibitor, Preferred Forms Glu, Met, Ala, Val, Pro, Asp,
Glu, Met, Ala, Gly, & Ala o, p treatment of Oral Tab/Cap 1-100
mg 2-80 mg 4-50 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr,
hypertension Oral Liquid 1-100 mg/ 2-80 mg/ 4-50 mg/5 ml Phe, Trp,
Hyp, Hsr, Car, Thr, Arg, Phe, Trp, 5 ml 5 ml Ort, Cav, Asn, Gln,
Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr,
and Sar Sirolimus All Doses expressed as drug base Lys, Leu, Ile,
Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Lys, b, h, i, j, k, l,
immunosuppressant Preferred Forms Glu, Met, Ala, Val, Pro, Asp,
Glu, Met, Ala, Gly, & Ala o, p in surgical human Oral Tab/Cap
0.1-20 mg 0.5-10 mg 1-8 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His,
Tyr, patients with Oral Liquid 0.1-20 mg/ 0.5-10 mg/ 1-8 mg/5 ml
Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, transplants; 5 ml 5 ml
Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and antibiotic; treating IM
Injections Tau, Djk, GABA, Cys, Ser, Hyp, Sar vitiligo psoriass,
Dcy, Thr, and Sar acne Tacrolimus All doses expressed as drug base
Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Ala, Thr, b,
c, g, h, i, immunosuppressant Preferred Forms Glu, Met, Ala, Val,
Pro, Asp, Glu, Met, Ala, Sar & Pro j, k, l, o, p in surgical
human Oral Tab/Cap 0.1-20 mg 0.2-15 mg 0.25-10 mg His, Tyr, Ser,
Nor, Arg, Val, Pro, His, Tyr, patients with Oral Liquid above/5 ml
above/5 ml above/5 ml Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp,
transplants; IV Infusions 1-20 mg/ml 2-15 mg/ml 2.5-8 mg/ml Ort,
Cav, Asn, Gln, Can, Gln, Asn, Cys and antibiotic; treating Tau,
Djk, GABA, Cys, Ser, Hyp, Sar vitiligo psoriass, Dcy, Thr, and Sar
acne Pimecrolimus All doses expressed as drug base Lys, Leu, Ile,
Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Ala, Thr, b, c, g, h, i,
immunosuppressant Preferred Forms Glu, Met, Ala, Val, Pro, Asp,
Glu, Met, Ala, Sar & Pro j, k, l, o, p in surgical human Oral
Tab/Cap 0.1-20 mg 0.2-15 mg 0.25-10 mg His, Tyr, Ser, Nor, Arg,
Val, Pro, His, Tyr, patients with Oral Liquid above/5 ml above/5 ml
above/5 ml Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp,
transplants; Ointment Cream 0.01-10% 0.1-5% 0.5-2% Ort, Cav, Asn,
Gln, Can, Gln, Asn, Cys and antibiotic treating Tau, Djk, GABA,
Cys, Ser, Hyp, Sar vitiligo psoriass, Dcy, Thr, and Sar acne
Clopidogrel All doses expressed as drug base Ser, Hyp, Thr, Lys, b,
c, h, i, j, treatment of Preferred Forms Ala, & Gly k, l, m, o,
p, q myocardial Oral Tab/Cap 10-250 mg 20-125 mg 25-100 mg 20-125
mg 25-100 mg infections Oral Liquid 10-250 mg/ 20-125 mg/ 25-100
mg/ 20-125 mg/5 ml 25-100 mg/5 ml 5 ml 5 ml 5 ml Amphotericin B All
doses expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu,
Ile, Gly, Ser, Hyp, Thr, Lys, b, c, g, i, j, treatment of fungus,
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Ala,
& Gly l, m, n, o, p, q expecially those IV Infusion 0.5-20
mg/kg/ 1-15 mg/kg/ 2-10 mg/kg/ His, Tyr, Ser, Nor, Arg, Val, Pro,
His, Tyr, acting on cell day day day Phe, Trp, Hyp, Hsr, Car, Thr,
Arg, Phe, Trp, membrane changing Topical Cream 0.01-10% 0.1-5%
0.5-2% Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and its permeability
Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Tenofovir All
doses expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu,
Ile, Gly, Lys, Gly, Ala, Pro, b, c, h, i, j, inhibitor of HIV
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Ser
& Sar k, l, o, p virus, treatment of Oral Tab/Cap 10-900 mg
50-750 mg 100-500 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr,
AIDS infections Oral Liquid 10-900 mg/ 50-750 mg/ 100-500 mg/ Phe,
Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, 5 ml 5 ml 5 ml Ort, Cav,
Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar
Dcy, Thr, and Sar Unoprostone All Doses expressed as drug base Lys,
Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Tyr, b, c,
h, i, j, treatment of Preferred Forms Glu, Met, Ala, Val, Pro, Asp,
Glu, Met, Ala, Pro, & Lys k, l, n, o, p, q glaucoma, Ocular
Drops 0.01-1% 0.05-0.5% 0.01-0.25% His, Tyr, Ser, Nor, Arg, Val,
Pro, His, Tyr, especially caused by Phe, Trp, Hyp, Hsr, Car, Thr,
Arg, Phe, Trp, age; lowers Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys
and intraocular pressure Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy,
Thr, and Sar Fulvestrant All doses expressed as drug base Lys, Leu,
Ile, Gly, Asp, Lys, Leu, Ile, Gly, Gly, Lys, Pro, Ala, B, c, g, j,
l, treating cancer, Preferred Forms Glu, Met, Ala, Val, Pro, Asp,
Glu, Met, Ala, Val & Sar o, p especially breast IM Injection
2-1250 mg/ 10-1000 mg/ 20-500 mg/ His, Tyr, Ser, Nor, Arg, Val,
Pro, His, Tyr, cancer 5 ml 5 ml 5 ml Phe, Trp, Hyp, Hsr, Car, Thr,
Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk,
GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Cefditoren All doses
expressed as drug base Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,
Ser, Hyp, Thr, Gly, b, c, h, i, j, antibiotics, Preferred Forms
Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Lys & Ala k, l, o,
p especially inhibits Oral Tab/Cap 20-500 mg 100-400 mg 150-300 mg
His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, H. influenzae; Oral
Liquid 20-500 mg/ 100-400 mg/ 150-300 mg/ Phe, Trp, Hyp, Hsr, Car,
Thr, Arg, Phe, Trp, Haemophilus para- 5 ml 5 ml 5 ml Ort, Cav, Asn,
Gln, Can, Gln, Asn, Cys and influenzae, Tau, Djk, GABA, Cys, Ser,
Hyp, Sar streptococcus, Dcy, Thr, and Sar Maraxella catarrhalis;
treatment of bronchitis, pharyngitis, tonsillitis, skin infections
Efavirenz All doses expressed as drug base Lys, Leu, Ile, Gly, Asp,
Lys, Leu, Ile, Gly, Gly, Lys, Pro, Ala, b, c, h, i, j, inhibitor of
HIV-1 Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala,
Sar, & Val k, l, o, p specific, non- Oral Tab/Cap 0.2-1.2 gm
300-800 mg 400-750 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr,
nucleoside, reverse Oral Liquid 0.2-1.2 gm/ 300-800 mg/ 400-750 mg/
Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, transcirptase; 5 ml 5
ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and treatment of
AIDS Tau, Djk, GABA, Cys, Ser, Hyp, Sar infections Dcy, Thr, and
Sar Eplerenone All doses expressed as drug base Lys, Leu, Ile, Gly,
Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Lys, b, c, h, i, j,
treatment of Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu,
Met, Ala, Gly & Val k, l, o, p hypertension, blocks Oral
Tab/Cap 10-250 mg 15-200 mg 20-150 mg His, Tyr, Ser, Nor, Arg, Val,
Pro, His, Tyr, binding of Oral Liquid 10-250 mg/ 15-200 mg/ 20-150
mg/ Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, aldosterone to 5
ml 5 ml 5 ml Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and
mineralo-corticoid Tau, Djk, GABA, Cys, Ser, Hyp, Sar receptors
Dcy, Thr, and Sar Treprostinil All Doses expressed as drug base
Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Gly, Lys, Val, Hyp, b,
c, g, h, i, Inhibits platelet Preferred Forms Glu, Met, Ala, Val,
Pro, Asp, Glu, Met, Ala, Thr & Ser j, k, l, o, p aggregation
and SC infusion 0.1-100 mg/ml 0.2-50 mg/ml 0.5-20 mg/ml His, Tyr,
Ser, Nor, Arg, Val, Pro, His, Tyr, vasodilation of Oral Tab/Cap
10-1000 mg 20-800 mg 25-500 mg Phe, Trp, Hyp, Hsr, Car, Thr, Arg,
Phe, Trp, systemic and Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and
pulmonary vascular Tau, Djk, GABA, Cys, Ser, Hyp, Sar bed,
treatment of Dcy, Thr, and Sar cardiovascular related conditions
Adefovir All doses expressed as drug base Lys, Leu, Ile, Gly, Asp,
Lys, Leu, Ile, Gly, Lys, Gly, Val, Ser, b, c, h, i, j, HIV reverse
Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Hyp,
& Pro k, l, o, p transcriptase Oral Tab/Cap 1-100 mg 2-50 mg
5-20 mg His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, inhibitors;
treatment Oral Liquid 1-100 mg/ 2-50 mg/ 5-20 mg/ Phe, Trp, Hyp,
Hsr, Car, Thr, Arg, Phe, Trp, of HIV infections 5 ml 5 ml 5 ml Ort,
Cav, Asn, Gln, Can, Gln, Asn, Cys and and AIDS Tau, Djk, GABA, Cys,
Ser, Hyp, Sar Dcy, Thr, and Sar
Above table is not exhaustive, and not final. Inventor has given
examples of certain drugs and proposed compostions and dosages, and
route of administration. However, above table is typical and does
not constraint the scope of this invention.
[0168] As used herein, the term "amino acid derivative" or "drug
derivative" and like terms refer to the drug bonded to an amino
acid residue. In addition, as used herein, the term L-amino acid
refers to an amino acid wherein the asymmetric carbon atom attached
to the amino and the carboxy group is in the L-configuration. For
purpose of this specification, it also includes glycine.
[0169] As indicated hereinabove, the amino acid derivatives of the
present invention have the same utility as the underlying drug
which is not bonded to the amino acid moiety. The amino acid
derivative of the present invention possesses at least one enhanced
quality identified hereinabove relative to the same drug without
the amino acid linkage.
[0170] Without wishing to be bound it is believe that the amino
acid derivatives of the present invention is the active agent in
vivo. Alternatively, the amino acid moiety may be cleaved off in
vivo, thereby making the active agent the drug itself in vivo.
Alternatively, a combination of both may occur in vivo. However,
whichever happens in vivo, when the amino acid derivative of the
present invention is administered to a patient, at least one of the
improved qualities enumerated hereinabove relative to the drug
without the amino acid linkage is realized.
[0171] The following non-limiting examples further illustrate the
invention:
Synthesis of Various Amino Acid Derivatives of Selected Drugs
I. Propofol Derivatives
[0172] Propofol (2,6-diisopropylphenol) is a low molecular weight
phenol which widely used as a central nervous system anesthetic,
and posses sedative and hypnotic activities. It is administered
intravenously in the induction and maintenance of anesthesia and/or
sedation in mammals. The major advantages of Propofol are that it
can induce anesthesia rapidly, minimal side effects and upon
withdrawal, the patient recovers quickly without prolonged
sedation.
Propofol
[0173] ##STR17##
[0174] Propofol has been shown to have a large number of
therapeutic applications, which are quite varying and somewhat
surprising. For example, it has been shown to be an effective
antioxidant, anti-emetic, anti-pruritic, anti-epileptic,
anti-inflammatory, and even seems to possess anti-cancer
properties.
Mechanism of Action:
[0175] The mechanism of action of Propofol has been extensively
studied. Its central nervous system anesthetic activity has been
shown to be related its high affinity for a specific subclass of
GABA receptors (Collins G. G. S., 1988, Br. J. Pharmacology. 542,
225-232). However, there are a number of different receptors in the
brain which are substrates for propofol, hence its varied
activities.
[0176] Propofol also has significant biological effect as an
antioxidant. Because of this generalized activity of propofol, it
is theoretically useful in the treatment of a number of
inflammatory processes where oxidation is an important factor. For
example, cyclooxygenase mediated prostaglandin synthesis results in
inflammation. By inhibiting oxidation in the respiratory tract, one
could use propofol in the treatment of acid aspiration,
adult/infant respiratory distress syndrome, airway obstructive
diseases, asthma, cancer and a number of other similar pathological
conditions.
[0177] Since oxidative tissue damage is a very common occurrence,
it has been suggested that propofol is useful in the treatment of
Parkinson's disease, Alzheimer disease, Friedrich's disease,
Huntington's disease, multiple sclerosis, amyotrophic lateral
sclerosis, spinal chord injuries, and various other
neurodegenerative diseases.
[0178] Propofol is currently available in the US market as an
intravenous emulsion marketed by Astra Zenaca under the brand name
Diprivan.RTM.. It is one the most widely used short acting central
nervous system anesthetics in the market. The concentration of
propofol is 10 mg/mL in non-pyrogenic sterile emulsion and the
formula contains soybean oil, glycerol, egg lecithin, disodium
edetate and sodium hydroxide.
[0179] A significant disadvantage of Propofol is that it is
completely insoluble in water. Even at very low concentrations of
10 mg/mL, the drug precipitates out of an aqueous solution in room
temperature. Therefore, manufacturers of this formulation use
heroic methods to emulsify this product in water using
extraordinarily complex and toxic emulsifying agents. For example,
manufacturers of the IV formulations use egg lecithin, Cremaphor
L.RTM., castor oil, and other similar emulsifiers.
[0180] However, use of such emulsifiers is associated with number
of problems. It is well known that various types of Cremaphor
L.RTM. emulsifiers can precipitate allergic reactions. Egg lecithin
and castor oil have been shown to produce anaphylactic shock in
some patients. Furthermore, maintenance of stability of propofol in
these emulsions is short lived and more expensive. Moreover, the
presence of egg lecithin and castor oil make the emulsion prone to
microbial growth. It may be possible to dissolve propofol in water
by complexing it with cyclodextrin, but cyclodextrin has not been
approved by the FDA for use in intravenous therapy.
[0181] Heretofore, no one has made a safe derivative of propofol.
The British patents 1,102,011, and 1,160,468 and U.S. Pat. No.
3,389,138 describe the various phenol esters of amino acids,
wherein the propofol is attached to a number of side chains which
when released in the body produce toxic effects.
[0182] U.S. Pat. No. 6,451,854 describe a number of substituted
alpha amino acetic acid esters of propofol, wherein propofol and
the side chain were substituted with a number of different chemical
groups. All the N,N-disubstituted glycine esters of propofol have
not shown to be non-toxic and many of the compounds described are
derivative of propofol. Thus when released in the body after the
cleavage of ester by the enzymes, many of the active drugs released
are not propofol, and hence they do not possesses any toxicity data
and are entirely new molecules with unknown therapeutic efficacy in
man.
[0183] In a published paper on the water soluble salts of amino
acid esters of the anesthetic agent propofol, (Int. J.
Pharmaceutics, 175[2]: 195-204, 1998) authors have synthesized a
number of water soluble derivatives of propofol. However, when
these derivatives are cleaved by esterase enzymes, substituted
non-natural amino acids with unknown toxicity profile are released
in the body.
[0184] Until now there has been no pharmaceutical preparation
available in the market that can deliver propofol without harmful
side effects. The present inventor has produced a number of water
soluble, non-toxic derivatives of propofol which are suitable for
delivering propofol in the body without any harmful side effects
and without the needs for toxic and expensive additives,
solubilizers and emulsifiers.
Accordingly, in one aspect, the present invention is directed to a
class of derivatives of Propofol. The derivative consists of the
carboxyl group of an amino acid esterified to the free hydroxyl
group present on the propofol molecules.
[0185] More specifically, one aspect of the present invention is
directed to, the compounds of the formula: ##STR18## or
pharmaceutically acceptable salts thereof; wherein AA is an amino
acid, in which the carboxyl group of AA is reacted with the
hydroxyl group of the Propofol.
[0186] In another aspect, the present invention is also directed to
a pharmaceutical composition comprising a therapeutically effective
amount of the various Propofol amino acid derivatives especially
Proline Ester drugs and a pharmaceutical carrier therefor.
Preferred amino acid derivatives are indicated in Table I.
[0187] In another embodiment, the present invention is directed to
a method of treating a patient in need of propofol therapy, which
method comprises administering to said patient an effective amount
of the Propofol.
[0188] In a further embodiment, the present invention is directed
to a method of enhancing the solubility of propofol in an aqueous
solution comprising reacting the hydroxyl functionality of the
Propofol with a amino acid, especially a naturally occurring amino
acid, and isolating the product thereof.
[0189] In a still further embodiment, the present invention is
directed to a method of substantially and in a therapeutically
efficacious manner, reducing or eliminating the potential toxic
side effects of current formulations containing toxic excipients
when administered to a patient which comprises reacting the
hydroxyl functionality of the propofol molecule with carboxyl
function of the amino acid, especially the preferred and most
preferred amino acids depicted in Table I, to form an ester
covalent bond respectively and isolating the product thereof and
administering said product to the patient.
[0190] Moreover, the inventor has discovered that when
unsubstituted naturally occurring amino acids are esterified to
propofol, the resulting derivatives are highly water soluble,
(>200 mg/L in water), release non-toxic amino acids upon
cleavage in the body and require none of the toxic emulsifier,
additives and other excipients.
[0191] Furthermore, the inventor has found that the amino acid
derivatives of propofol of the present invention are highly
effective central nervous system anesthetics. Thus the current
amino acid derivatives are effective central nervous system
anesthetics, with or without releasing the active parent drug.
[0192] The amino acid esters of the present invention are at least
10 times more soluble that propofol in water in room temperature.
Especially the glycine, proline and lysine esters of propofol are
soluble at the range of more than 100 mg/ml, and in case of lysine
it is greater than 250 mg/mL.
[0193] The amino acid derivatives of the present invention are not
expected to possess any antioxidant activity due to blockage of the
phenolic group responsible for such; however the present inventor
has found that the amino acid derivatives of propofol are effective
anesthetics with or without releasing propofol. Further, the
Propofol Proline Ester drugs described, when administered in vivo,
maintains its pharmacological and anti-oxidant properties.
[0194] The amino acid derivative of propofol of the present
invention clearly provides a number of advantages over propofol,
for example, all of the side chains cleaved from these derivatives
are naturally occurring essential amino acids and hence are
non-toxic. This results in high therapeutic index. Secondly the
derivatives are readily cleaved in the body to release propofol,
however, if not cleaved the amino acid derivatives exhibit the same
utility. Furthermore, due to their high water solubility, they can
be easily administered by either forming an in-situ solution just
before IV administration using lyophilized sterile powder or
providing the drug in solution in prefilled syringe or bottles for
infusion. The amino acid esters are more stable than propofol since
OH group in propofol is blocked thereby prevents oxidation. For
example, the Propofol Proline Ester drugs of the present invention
are more effective then propofol itself without the toxicity and
other pharmaceutical problems associated with current marketed
formulations.
[0195] The amino acid derivatives of propofol of the present
invention possess anti-inflammatory, anti-oxidant, anti-cancer,
anti-convulsive, anti-emetic and anti-pruritic properties.
[0196] These derivatives of propofol of the present invention are
effective in treating diseases or conditions in which Propofol
normally are used. The derivatives disclosed herein may or may not
be transformed within the body to release the active compound, and
can enhance the therapeutic benefits of the Propofol moiety by
reducing or eliminating biopharmaceutical and pharmacokenetic
barriers associated with each of them. However it should be noted
that these derivatives themselves will have sufficient activity
without releasing any active drug in the mammals. Since the
derivatives are more soluble in water then Propofol, it does not
need to be associated with a carrier vehicle, such as alcohol or
castor oil which may be toxic or produce unwanted side reactions.
Moreover, oral formulations containing the derivatives of Propofol
are absorbed into the blood and are quite effective.
[0197] Thus, the amino acid derivative of the present invention,
e.g., the amino acid derivative of propofol, enhances the
therapeutic benefits by removing biopharmaceutical and
pharmacokenetic barriers of existing drugs.
[0198] Furthermore, the amino acid derivatives, e.g., the amino
acid derivatives of propofol, are easily synthesized in high yields
using reagents which are readily and commercially available.
Overview:
[0199] The procedure for the synthesis of the glycine, L-proline,
and L-lysine esters of Propofol is depicted hereinbelow. However,
these are exemplary and any amino acid derivative thereof can be
prepared using the following methodology. The complete procedure
and analytical data is given in the Experimental Section. In
general, as shown in the following scheme Propofol (10 g) was
coupled with the N-Boc protected amino acid (1 equivalent) in the
presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,
hydrochloride (EDC) in the presence of a catalytic amount of
4-(N,N-dimethyamino)-pyridine (DMAP). The EDC was removed by
extraction with water. After drying over sodium sulfate,
filtration, and concentration thereof, the crude protected amino
acid ester of Propofol was purified by flash chromatography to
generate the protected esters in 50-60% yield. The protecting
groups were then removed by stirring the protected esters in
diethyl ether saturated with hydrochloric acid (gas) at room
temperature. Yields for the deprotection step were generally
60-95%. After filtration and drying the hydrochloride salts of the
glycine and L-proline esters of Propofol did not require additional
purification. The hydrochloride salt of the L-lysine-Propofol ester
was crystallized once from ethanol to remove a trace of
mono-protected L-lysine-Propofol ester. Synthetic Sequence:
##STR19## ##STR20## Synthesis of the Glycine, L-proline, and
L-lysine Esters of Propofol: a) EDC, DMAP, CH.sub.2Cl.sub.2; b) HCl
(g), Et.sub.2O. Experimental Section:
[0200] The synthesis of SPI0010, SPI0011 and SPI0013 were conducted
in batches. Generally a small-scale experiment was performed first
followed by a larger batch. Reagents mentioned in the experimental
section were purchased at the highest obtainable purity from
Aldrich, Acros, or Bachem, except for solvents, which were
purchased from either Fisher Scientific or Mallinkrodt.
1) SPI0010
[0201] Propofol (9.98 g, 55.97 mmole) was dissolved in
dichloromethane (200 mL) at room temperature, under an argon
atmosphere. N-t-Butyloxocarbonyl-glycine (11.2 g, 63.91 mmole) was
added along with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,
hydrochloride (EDC, 11.1 g, 57.9 mmole) and
4-(N,N-dimethylamino)-pyridine (DMAP, 1.5 g, 12.27 mmole). After
stirring for 21 hours under an argon atmosphere at room
temperature, water (200 mL) was added and the layers were
separated. The dichloromethane layer was washed with water (200 mL)
and dried for 1 hour over sodium sulfate (5 g). After filtration
and concentration under reduced pressure, the remaining oil was
purified by flash chromatography on silica gel (250 g), eluting
with hexanes/ethyl acetate (10:1). The procedure generated the
protected N-BOC protected glycine ester of Propofol as a white
solid (11.34 g, 60% yield). ##STR21##
tert-Butoxycarbonylamino-acetic acid 2,6-diisopropylphenyl
ester
[0202] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.25-7.13 (m,
3H), 5.18 (br s, 1H), 4.22 (d, 2H, J=5.7 Hz), 2.89 (m, 2H), 1.46
(s, 9H), 1.18 (d, 12H, J=6.9 Hz).
[0203] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=169.35, 155.75,
145.22, 140.35, 126.90, 124.14, 80.32, 42.66, 28.54, 27.79,
23.57.
[0204] The Propofol-Boc-gycine ester (11.28 g, 33.6 mmole) was
dissolved in anhydrous diethyl ether (200 mL) at room temperature.
Hydrochloric acid (gas) was passed through the solution for 45
minutes while stirring. The mixture was allowed to stir at room
temperature for 48 hours under an argon atmosphere. After 48 hours
hexanes (200 mL) were added and the precipitate was filtered. The
white solid was dried under high vacuum for 5 hours at 88.degree.
C. The experiment produced SPI0010 (8.73 g, 95% yield, purity 99.9%
by HPLC) as a white solid. ##STR22##
Amino-acetic acid 2,6-diisopropyl-phenyl ester, hydrochloride
[0205] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=8.77 (br s, 3H),
7.20-7.08 (m, 3H), 4.14 (m, 2H), 2.87 (m, 2H), 1.11 (d, 12H, J=7
Hz).
[0206] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=166.42, 144.84,
140.42, 127.10, 124.06, 40.47, 27.61, 23.55.
[0207] CHN analysis:
[0208] calc.: C, 61.87; H, 8.16; N, 5.15. found: C, 61.14; H, 8.20;
N, 5.14.
2) SPI0011
[0209] Propofol (10.03 g, 56.23 mmole) was dissolved in
dichloromethane (100 mL) at room temperature, under an argon
atmosphere. N-t-Butyloxocarbonyl-L-proline (14.04 g, 65.22 mmole)
was added along with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,
hydrochloride (EDC, 11.95 g, 62.33 mmole) and
4-(N,N-dimethylamino)-pyridine (DMAP, 1.1 g, 9.0 mmole). After
stirring for 3 hours under an argon atmosphere at room temperature,
water (100 mL) was added and the layers were separated. The
dichloromethane layer was washed again with water (100 mL) and
dried for 1 hour over sodium sulfate (5 g). After filtration and
concentration under reduced pressure, the remaining oil was
purified by flash chromatography on silica gel (250 g), eluting
with hexanes/ethyl acetate (10:1). The procedure generated the
protected N-BOC protected L-proline ester of Propofol as a clear
oil (11.34 g, 66% yield) that solidified on standing in the
freezer. ##STR23##
Pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester
2-(2,6-diisopropyl-phenyl)ester
[0210] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.31-7.20 (m,
3H), 4.73 (m, 1H), 3.70-3.50 (m, 2H), 3.20-2.94 (m, 2H), 2.46-2.20
(m, 2H), 2.20-2.0 (m, 2H), 1.55 (m, 9H), 1.25 (m, 12H).
[0211] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=171.87, 171.01,
154.34, 153.93, 145.35 145.23, 140.06, 140.21, 126.69, 126.53,
123.95, 80.28, 79.89, 59.14, 46.67, 46.42, 31.10, 30.17, 28.61,
28.56, 28.56, 27.44, 27.18, 23.47.
[0212] The Propofol-Boc-L-proline ester (13.95 g, 37.14 mmole) was
dissolved in anhydrous diethyl ether (100 mL) at room temperature.
Hydrochloric acid (gas) was passed through the solution for 60
minutes while stirring. The mixture was allowed to stir at room
temperature for 22 hours under an argon atmosphere. After 22 hours,
hexane (50 mL) was added and the precipitate was filtered. The
white solid was dried under high vacuum for 5 hours at 88.degree.
C. The experiment produced SPI0011 (9.1 g, 81% yield, purity 99.1%
by HPLC) as a white solid. ##STR24##
Pyrrolidine-2(S)-carboxylic acid 2,6-diisopropyl-phenyl ester,
hydrochloride
[0213] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=10.15 (br s, 2H),
7.27-7.14 (m, 3H), 4.78 (t, 1H, J=7.8 Hz), 3.56 (m, 2H), 2.85 (m,
2H), 2.64 (m, 1H), 2.40 (m, 1H), 2.20 (m, 1H), 2.05 (m, 1H), 1.18
(m, 12H).
[0214] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=168.30, 144.23,
139.74, 126.98, 123.96, 51.58, 38.21, 29.32, 26.64, 26.18, 23.71,
23.02, 21.67.
[0215] CHN analysis:
[0216] calc.: C, 65.48; H, 8.40; N, 4.49. found: C, 65.50; H, 8.43;
N, 4.50.
3) SPI0013
[0217] The dicyclohexylamine salt of di-N-boc-L-lysine (23.62 g,
0.0447 mole) was added to diethyl ether (200 mL) and potassium
hydrogen sulfate (9.14 g) in water (200 mL) that was cooled in an
ice/water bath. After strirring for 20 minutes, the layers were
separated. The ether layer was extracted three times with cold
water (100 mL). The ether layer was then dried over sodium sulfate
(15 g) for one hour, filtered, and concentrated under reduced
pressure. The procedure generated the free acid of
N,N'-di-boc-L-lysine (15.5 g, 100% recovery).
[0218] Propofol (8.0 g, 45 mmole) was dissolved in dichloromethane
(100 mL) at room temperature, under an argon atmosphere.
N,N'-di-t-Butyloxocarbonyl-L-lysine (15.5 g, 44.7 mmole) was added
along with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,
hydrochloride (EDC, 8.62 g, 45 mmole) and
4-(N,N-dimethylamino)-pyridine (DMAP, 0.55 g, 4.5 mmole). After
stirring for 3 hours under an argon atmosphere at room temperature,
water (100 mL) was added and the layers were separated. The
dichloromethane layer was washed again with water (100 mL) and
dried for 1 hour over sodium sulfate (5 g). After filtration and
concentration under reduced pressure, the remaining oil was
purified by flash chromatography on silica gel (250 g), eluting
with hexanes/ethyl acetate (9:1). The procedure generated the
protected N-BOC protected L-lysine ester of Propofol as a white
foam (12.42 g, 55% yield). ##STR25##
2(S),6-Bis-t-butoxycarbonylamino-hexanoic acid
2,6-diisopropyl-phenyl ester
[0219] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.28-7.15 (m,
3H), 5.22 (d, 1H, J=8.4 Hz), 4.70 (m, 1H), 4.59 (m, 1H), 3.17 (m,
2H), 2.93 (m, 2H), 2.09 (m, 1H), 1.86 (m, 1H), 1.67-1.54 (m, 4H),
1.48 (s, 9H), 1.46 (s, 9H), 1.20 (m, 12H).
[0220] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=171.82, 156.10,
155.65, 145.25, 140.30, 126.80, 124.03, 80.14, 79.28, 53.76, 40.29,
32.09, 28.66, 28.54, 27.48, 23.91, 23.10.
[0221] The Propofol-di-Boc-L-lysine ester (12.34 g, 24.37 mmole)
was dissolved in anhydrous diethyl ether (250 mL) at room
temperature. Hydrochloric acid (gas) was passed through the
solution for 60 minutes while stirring and cooling in an ice/water
bath. The mixture was allowed to stir at room temperature for 48
hours under an argon atmosphere. After 48 hours the precipitate was
filtered and crystallized from ethanol (100 mL). The white solid
was dried under high vacuum for 4 hours at 90.degree. C. The
experiment produced SPI0013 (5.5 g, 60% yield, purity 98.6% by
HPLC) as a white solid. ##STR26##
2(S),6-Diamino-hexanoic acid 2,6-diisopropyl-phenyl ester,
dihydrochloride
[0222] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=9.05 (br s, 3H),
8.35 (br s, 3H), 7.26-7.13 (m, 3H), 4.43 (t, 1H, J=6 Hz), 3.0-2.6
(m, 4H), 2.09 (m, 2H), 1.80-1.50 (m, 4H), 1.10 (d, 12H, J=7
Hz).
[0223] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=168.30, 144.23,
139.74, 126.98, 123.96, 51.58, 38.21, 29.32, 26.64, 23.71, 23.02,
21.67.
[0224] CHN analysis:
[0225] calc.: C, 56.99; H, 8.50; N, 7.38. found: C, 56.48; H, 8.56;
N, 7.30.
Anasthetic Effect in Male Albino Mice for Derivatives of
Propofol
[0226] The present study was conducted to evaluate the efficacy of
new formulations of propofol injection using righting reflex as an
index in albino mouse. Propofol injection served as reference
control. Male albino mice were exposed to propofol injection and to
3 new formulations of propofol at 3 dose levels--low, intermediate
and high dose levels. However the doses of low, intermediate and
high for different formulations and reference control were
different.
[0227] The different doses for each formulation and reference
control were selected based on the recommendation of Sponsor. All
the doses were expressed as propofol molar equivalents. The doses
used for the main experiment for different formulations and
reference control were presented below. TABLE-US-00003 TABLE 2 Low
Dose Intermediate High Dose Test Item (mg/kg) Dose (mg/kg) (mg/kg)
Propofol Lysine Ester 40.0 45.0 50.0 Propofol Glycine Ester 30.0
35.0 40.0 Propofol Proline Ester 25.0 30.0 35.0 Propofol Injection
10.0 15.0 20.0 (Reference control)
Efficacy (Anesthetic Property)
[0228] The efficacy in terms of time required to regain the
righting reflex at different dose levels--low, intermediate and
high dose for different formulations and propofol injection are
presented below. TABLE-US-00004 TABLE 3 Low Dose Intermediate Dose
High Dose Formulation 1 0.00 .+-. 0.00 0.00 .+-. 0.00 546.00 .+-.
0.00 Formulation 2 532.10 .+-. 217.61 299.80 .+-. 211.32 641.00
.+-. 137.86 Formulation 3 282.00 .+-. 162.09 421.70 .+-. 112.92
237.25 .+-. 197.47 Propofol 20.20 .+-. 43.42 123.40 .+-. 33.70
177.70 .+-. 70.31 injection (Reference control) All doses produced
statistically significant results at p < 0.05.
[0229] All doses produced statistically significant results at
p<0.05.
Low Dose
[0230] Propofol Glycine Esterl was found to be more effective
followed by formulation 3 and reference control. Formulation 1 did
not show any anesthetic effect.
[0231] No mortality was observed at low dose level with different
formulations or propofol injection.
Intermediate Dose
[0232] Propofol Proline Ester ester was found to be more effective
followed by Propofol Glycine Esterl and reference control. Propofol
lysine ester did not show any anesthetic effect.
High Dose
[0233] Propofol Glycine Esterl was found to be more effective
followed by propofol lysine ester, Propofol Proline Esterraline
ester and reference control.
Dose Response Relation
Propofol Lysine Ester
[0234] The statistical analysis showed a significant improvement at
5% significance level in the relative efficacy for the high dose
when compared to low dose group.
Propofol Glycine Ester
[0235] The responses to the dose levels were not statistically
evident from the analysis, though the comparisons showed
significance at 5% level for mid dose to low dose.
Propofol Proline Ester
[0236] No significant dose--response effects were evident from the
statistical analysis.
Propofol Injection (Reference Control)
[0237] For reference control group, the dose response effect were
statistically significant and clearly evident.
[0238] In conclusion, it was observed that based on the time
required to regain the righting reflex, Propofol Glycine Ester and
the lysine ester have relatively better efficacy than the other two
derivatives (Propofol Proline ester and the drug) in inducing
anesthesia in albino mice.
14-Day Chronic Toxicity Studies in Albino Mice Using Propofol
Glycine Ester and Proline Ester:
[0239] The present study was conducted to evaluate the relative
safety of the two new formulations of Propofol injection
[0240] Propofol Glycine Ester
[0241] Propofol Proline Ester
by conducting a 14 day repeated dose toxicity study through
intravenous route using albino mice as test system. Propofol
injection served as reference control.
[0242] Propofol and its derivatives above were administered to
albino mice (Swiss strain), through intravenous route daily for a
period of 14 days using sterile sodium chloride solution as
vehicle. The study was conducted at one dose level only along with
a vehicle control group as per the recommendation of the
Sponsor.
[0243] The doses used in the present study for the two amino acid
ester and Propofol injection were different and presented below.
The doses were selected as per the recommendation of the Sponsor.
The test doses are expressed as Propofol molar equivalents.
TABLE-US-00005 TABLE 4 Dose Equivalent [in terms of weight of the
Propofol] Test item Test Item Group (mg/kg) [mg/kg] Vehicle Vehicle
Control 0.0 0.0 (Propofol Glycine Ester) Test Group 1 30.0 45.60
(Propofol Proline Test group 2 25.0 43.75 Ester) Propofol injection
Reference Control 15.0 15.00
[0244] The salient features of the study are as follows:
1. All the animals of control and the test group 1 (Propofol
Glycine Ester), test group 2 (Propofol Proline Ester) and reference
control group (Propofol Injection) survived through the dosing
period of 14 days.
[0245] 2. None of the animals of the vehicle control and the test
group 1 (Propofol Glycine Ester), test group 2 (Propofol Proline
Ester) and Reference Control (Propofol Injection) group exhibited
any clinical symptoms of toxicity. However animals dosed with the
amino acid esters of Propofol or reference control showed
anesthetic effect immediately after dosing. This effect was
attributed to the pharmacological property of the amino acid esters
or reference control.
[0246] 3. Body weight gain of animals of test group 2 (Propofol
Proline Ester) and reference control (Propofol Injection) was found
to be normal and comparable to that of vehicle control group of
animals. However, the body weight gain was reduced in both the
sexes of test group 1 (Propofol Glycine Ester). Males and females
of test group 1 (Propofol Glycine Ester) showed a reduced body
weight gain of 25.87% and 26.94% respectively compared to vehicle
control group animals.
4. Feed intake of the animals of test group 1 (Propofol Glycine
Ester), test group 2 (Propofol Proline Ester) and reference control
(Propofol injection) was found to be normal and comparable to that
of vehicle control group.
5. Results of hematological analysis revealed the following changes
in the animals of different groups when compared to reference
control.
Propofol Glycine Ester
[0247] The granulocytes counts significantly increased [p<0.05]
in female animals of Propofol Glycine Ester when compared to the
female animals of Propofol injection (RC).
[0248] The MCV significantly decreased [p<0.05] in female
animals of Propofol Glycine Ester when compared to the female
animals of Propofol injection (RC).
Propofol Proline Ester
[0249] The granulocytes significantly decreased [p<0.05] in male
animals of Propofol Proline Ester when compared to the male animals
of Propofol injection (RC).
[0250] The lymphocytes counts significantly increased [p<0.05]
in male animals of Propofol Proline Ester when compared to the male
animals of Propofol injection (RC).
[0251] The MCV significantly decreased [p<0.05] in female
animals of Propofol Proline Ester when compared to the female
animals of Propofol injection (RC).
Vehicle Control
[0252] The lymphocytes counts significantly increased [p<0.05]
in male and female animals of vehicle control when compared to the
male and female animals of Propofol injection (RC).
6. Results of clinical chemistry analysis revealed the following
changes in the animals of different groups when compared to
reference control.
Propofol Proline Ester
[0253] The level of urea significantly decreased [p<0.05] in
female animals of Propofol Proline Ester when compared to the
female animals of Propofol injection (RC). The level of sodium
significantly increased [p<0.05] in male animals of Propofol
Proline Ester when compared to the male animals of Propofol
injection (RC).
Vehicle Control
[0254] The cholesterol level significantly decreased [p<0.05] in
female animals of vehicle control when compared to the female
animals of Propofol injection (RC).
[0255] Thus the results the present study showed that the [0256]
behavior of animals, [0257] body weight gain (except in male and
female animals of Propofol Glycine Ester) [0258] feed consumption,
[0259] Organ weights (absolute and relative) [0260] histopathology
of different organs of the animals treated with Propofol Glycine
Ester and Propofol Proline Ester were found to be normal and
comparable to that of the animals of the reference group. II. Amino
Acid Derivatives of Non-Steroidal Anti-Inflammatory Drugs
(NSAIDs)
[0261] The NSAIDs comprise a class of structurally distinctive
carboxylic acid moiety attached to a planar aromatic functionality.
Examples include: acetyl salicyclic acid, salicyclic acid,
diflunisal, ibuprofen, fenoprofen, carprofen, flurbiprofen,
ketoprofen, naproxen, sulindac, indomethacin, etodolac, tolmetin,
ketorolac, diclofenac, and meclofenarnate. The NSADIs posess
anti-inflammatory, analgesic, antipyretic and anti-clotting
activity.
[0262] Examples of the chemical structures of this uniques class of
compounds showing wide variety of pharmacological activities are
shown below. ##STR27## ##STR28## NSAIDs are widely used for the
treatment of acute and chronic pain, management of edema, tissue
damage resulting from inflammatory joint diseases and also,
effective anti-clotting agents in the treatment of myocardial
infraction. A number of the agents also possess antipyretic
activity in addition to analgesic and anti-inflammatory action,
thus useful in reducing fever.
[0263] Some drugs in the above group have also been prescribed for
Rheumatoid Arthritis, Osteoarthritis, acute gout, ankolysing
spondylitis, and dysmenorrhea.
Mechanism of Action:
[0264] The major mechanism by which the NSAIDs produce their
therapeutic effect is via inhibition of prostaglandin synthesis.
Specifically NSAIDs inhibit cyclooxygenases, such as COX-1 and
COX-2 enzymes, where these two enzymes are responsible for
synthesis of prostaglandins. While COX-1 enzyme is important for
the regulation of platelet aggregation, regulation of blood flow in
kidney and stomach, and regulation of gastric acid secretion, COX-2
enzyme plays an important role in the pain and inflammatory
processes. NSAIDs significantly increase clotting time and can be
used for prophylaxis of thromboembolism and myocardial
infarction.
[0265] All NSAIDs are relatively medium to strong organic acids
with pKa's in the 3-6 range. Most of them are carboxylic acid
derivatives. Acidic group is essential for COX inhibitory activity
and in physiological pH, all the NSAIDs are ionized. All of them
have quite varying hydrophilic lipophilic balance, and these are
functions of their aromatic and aliphatic side chains and other
heterocyclic variations in their structures. Most of the NSAIDs are
highly bound to plasma proteins and often competitively replace
other drugs which have similar affinity for plasma proteins. Hence
concomitant administration of NSAIDs with other therapeutic class
of drugs must be carefully evaluated to prevent drug interactions.
Most of the drugs, due to their acidic carboxyl group, are
metabolized by the mammals in vivo. The major pathway of metabolic
clearance of a number of NSAIDs is glucuronidation followed by
renal elimination.
[0266] Use of acetylsalicylic acid (aspirin) in the prophylaxis of
coronary heart diseases is now well known, and this drug has proved
to be a lifesaver for a number of patients with myocardial
infarction (Heart Attacks). Several additional uses have already
been documented for aspirin, for example, it was recently reported
in the medical journal Lancet (Vol 349, p 1641) that aspirin
reduces the risk of stroke in patients with early warning signs of
transient ischemic heart attacks. Pre-eclampsia and fetal growth
retardation, both caused by blockages of the blood vessels of the
placenta, are two of the commonest complications of
pregnancy--there are millions of cases of pre-eclampsia in the
world each year. In a trial involving more than 9000 women in 16
countries, a daily dose of 60 mg aspirin reduced the risk of
pre-eclampsia by 13 percent. (Aspirin Foundation website). Aspirin
has also been shown to be effective in some studies to prevent
colon cancer, lung cancer and pancreatic cancer in post-menopausal
women. Since aspirin can improve blood flow, its usefulness in the
treatment of diabetes and certain forms of dementia such as
Alzheimer's disease are becoming increasingly clear.
[0267] Because of their unique pharmaceutical potential, the NSAIDs
have attracted considerable attention in the press. The primary
area of clinical investigation for the above drugs has been as
non-steroidal anti-inflammatory agents, in particular in relation
to their application to treat patients suffering from pain,
arthritis, (Rheumatoid and Osteo) other inflammatory reactions,
fever and for the prophylaxis of coronary heart diseases. These
drugs are also used in the treatment of migraine headaches,
menstrual syndromes, back pain and gout.
[0268] Despite the very major contribution which NSAIDs have made,
difficulties have been encountered in providing more effective and
convenient means of administration (e.g., galenic formulations, for
example, oral dosage form, which are both convenient and for the
patient as well as providing appropriate bioavailability and
allowing dosaging at an appropriate and controlled dosage rate). In
addition, there are reported occurrences of undesirable in vivo
side reactions associated with NSAIDs; in particular severe gastric
and duodenal ulcers, mucosal erythema, and edema, erosions,
perforations, blood in stool, ulcerative colitis have been obvious
serious impediments to their wider use or application. The dual
injury theory involves NSAID-mediated direct damage, followed by a
systemic effect in which prostaglandin synthesis is inhibited.
Topical injury may also occur as a result of the biliary excretion
of active hepatic metabolites and subsequent duodenogastric reflux.
(Arthritis and Rheumatism 1995; 38(1):5-18) The effects are
additive; either topical or systemic mechanisms alone are
sufficient to produce gastro duodenal mucosal damage. Recently,
many of the COX-2 inhibitory NSAIDs such as Vioxx.RTM. and
Celebrex.RTM. have been removed from the market due to their
undesirable and life threatening side effects.
[0269] Moreover, the above mentioned NSAIDs are characteristically
highly hydrophobic and readily precipitate in the presence of even
very minor amounts of water, e.g., on contact with the body (e.g.,
stomach fluids). It is accordingly extremely difficult to provide
e.g., oral formulations which are acceptable to the patient in
terms of form and taste, which are stable on storage and which can
be administered on a regular basis to provide suitable and
controlling patient dosaging.
[0270] Proposed liquid formulations, e.g., for oral administration
of NSAIDs, have heretofore been based primarily on the use of
natural gums, like Xanthan, cellulose, citric acid, and lime flavor
etc. See e.g., U.S. Pat. No. 5,780,046. Commercially available
NSAIDs drink-solution employs incompatible orange color and berry
flavor, citric acid, Xanthan Gum, polysorbate 80, pregelatinized
starch, glycerin, sodium benzoate, and additional artificial colors
and flavors. Use of the drink solution and similar composition as
proposed in the art is however accompanied by a variety of
difficulties.
[0271] Further, the palatability of the known oil based system has
proved problematic. The taste of the known drink-solution is, in
particular, unpleasant. Admixture with an appropriate flavored
drink, for example, chocolate drink preparation, at high dilution
immediately prior to ingestion has generally been practiced in
order to make regular therapy at all acceptable. Adoption of oil
based systems has also required the use of high ethanol
concentrations which in and of itself is inherently undesirable, in
particular where administration to children is forseen. In
addition, evaporation of the ethanol, e.g., from capsules (adopted
in large part, to meet problems of palatability, as discussed or
other forms (e.g., when opened) results in the development of a
NSAID precipitate. Where such compositions are presented in, for
example, soft gelatin encapsulated form presents particular
difficulty which necessitates packaging of the encapsulated product
in an air-tight component, for example, an air-tight blister or
aluminum-foil blister package. This in turn renders the product
both bulky and more expensive to produce. The storage
characteristics of the aforesaid formulations are, in addition, far
from ideal.
[0272] Gastric irritability of the NSAIDs has been a topic of great
concern to the practicing physicians as well as patients. Acute
uses of aspirin, fenoprofen, flurbiprofen, indomethacin, ketorolac,
meclofenamate, mefanamic acid, and piroxicam produce serious GI
side effects. Even Ibuprofen has been shown to cause severe gastric
lesions upon long term use. Gastrointestinal toxicity is the most
frequently encountered side effect associated with NSAIDs and
presents considerable concern. Approximately one half of all
hospital admissions for a bleeding ulcer are attributed to the use
of NSAIDs, aspirin, or the two taken in combination during the week
prior to admission. (Faulkner G, Prichard P, Somerville K, et al.
Aspirin and bleeding peptic ulcers in the elderly. Br Med J. 1988;
297:1311-1313). A survey of Tennessee Medicaid patients who were
hospitalized with GI complications showed that patients who used
NSAIDs had approximately a fourfold greater risk for developing GI
hemorrhage or peptic ulcer disease than patients not taking NSAIDs.
(Griffin M R, Piper J M, Daugherty J R, et al. Nonsteroidal
anti-inflammatory drug use and increased risk for peptic ulcer
disease in elderly persons. Ann Intern Med. 1991; 114:257-263).
Serious GI events, according to the FDA, occur in as many as 2% to
4% of patients per year who are taking continuous NSAID therapy for
rheumatoid arthritis. The relative risk of gastric ulcer (4.725),
duodenal ulcer (1.1 to 1.6), bleeding (3.8), perforation, and death
are all increased by NSAID use when such patients are compared to
those who do not take these products. In 1989, patients with
rheumatoid arthritis had approximately 20,000 hospitalizations per
year with an estimated cost of $ 10,000 per stay. (Fries J F,
Miller S R, Spitz P W, et al. Toward an epidemiology of gastropathy
associated with nonsteroidal anti-inflammatory drug use. J
Gastroenterology. 1989; 96:647-655).
[0273] There is also a need for providing some of the NSAIDs in a
water soluble form for injection. It is well known that high
concentrations of alcohol and tromethamine used to form a salt in
the current formulations of Ketorolac are toxic. At present there
is no formulation that would allow the NSAIDs to be in aqueous
solution at the concentrations needed due to poor water solubility
of the drug.
[0274] Beyond all these very evident practical difficulties lies
the occurrence of undesirable side reactions already alluded to,
observed employing available oral dosage forms.
[0275] Several proposals to meet these various problems have been
suggested in the art, including both solid and liquid oral dosage
forms. An overriding difficulty which has however remained is the
inherent insolubility of the NSAIDs in aqueous media, hence
preventing the use of a dosage form which can contain NSAIDs in
sufficiently high concentration to permit convenient use and yet
meet the required criteria in terms of bioavailability, e.g.
enabling effective resorption from the stomach or gut lumen and
achievement of consistent and appropriately high blood/blood-serum
levels.
[0276] The present derivatives of NSAIDs overcome the problems
described hereinabove. More specifically, an embodiment of the
present invention is directed to a derivative of NSAID which
significantly enhances its solubility in aqueous solutions, thereby
avoiding the need to utilize a carrier, such as ethanol or castor
oil when administered as a solution. Moreover, the derivatives of
NSAID, in accordance with the present invention, do not exhibit the
side effects of the prior art formulations. Further, the
derivatives of the present invention are almost completely devoid
of gastric irritability upon oral administration, thereby enhancing
significantly the therapeutic index of the derivatives tested and
their efficacy.
[0277] Accordingly, in one aspect, the present invention is
directed to a derivative of NSAIDs.
[0278] The preferred derivatives of the NSAIDs have the formula
##STR29## ##STR30## or pharmaceutically acceptable salts thereof;
wherein Y is either NH-AA or O-AA and AA is an amino acid residue,
in which either an amine group on the main chain or for those basic
acids, on the side chain, if one is present (and more preferably on
the main chain), or the hydroxyl group on the side chain of the AA
acid residue is reacted with the carboxylic acid group of the
NSAIDs.
[0279] The present invention is also directed to a pharmaceutical
composition comprising a therapeutically effective amount of the
various amino acid derivatives of the NSAIDs above and a
pharmaceutical carrier therefor.
[0280] In another embodiment, the present invention is directed to
a method of treating a patient in need of NSAID therapy, which
method comprises administering to said patient an effective amount
of the amino acid derivatives of the NSAIDs of the present
invention.
[0281] In a further embodiment, the present invention is directed
to a method of enhancing the solubility of NSAID in an aqueous
solution comprising reacting the carboxyl functionality of each of
the NSAID with an amino acid under effective conditions to form a
covalent bond between the amino acid and the NSAID and isolating
the product thereof.
[0282] In a still further embodiment, the present invention is
directed to a method of substantially and in a therapeutically
efficacious manner, reducing or eliminating the gastric mucosal
damage of NSAIDs when administered to a patient which comprises
reacting the carboxyl functionality of each of the NSAID molecule
with either the amine functionality on the amino acid or hydroxyl
function on the amino acids having a hydroxy group on the side
chain, e.g., Threonine, hydroxy proline, tyrosine, serine and the
like, or the amine of those amino acids having an amino group on
the side chain, to form either an amide or ester or an amide
covalent bond, respectively, and isolating the product thereof and
administering said product to the patient. Preferably, the carboxyl
functionality of the NSAID is reacted with the amine functionality
on the main chain and/or the hydroxy group on the side chains of
those amino acids having a hydroxy group on the side chain.
A. Synthesis of Ibuprofen Amino Acid Derivatives
Overview:
[0283] The procedure for the synthesis of the L-serine,
L-threonine, and L-hydroxyproline esters of Ibuprofen is outlined
in Synthetic Sequence section. The complete procedure and
analytical data is given in the Experimental Section. Again, these
synthetic schemes are exemplary. The scheme is applicable for other
amino acids in the preparation of the NSAID derivatives of the
present invention. In general, (.+-.)-Ibuprofen (4-10 g, in
batches) was coupled with the N-benzyloxy/benzyl ester protected
amino acids (1 equivalent) with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,
1 equivalent) in the presence of a catalytic amount of
4-(N,N-dimethyamino)-pyridine (DMAP). Once the reactions were
complete, any excess EDC was removed by extraction with water, DMAP
was removed by extraction with dilute acid, and Ibuprofen was
removed by extraction with sodium bicarbonate. After drying over
sodium sulfate, filtration, and concentration the crude protected
amino acid esters of (.+-.)-Ibuprofen were either used directly or
purified by flash chromatography on silica gel to generate the
protected esters in good yield (85-95%). The column chromatography
was generally not necessary if a slight excess of Ibuprofen and
coupling agent were used, and a thorough extraction procedure was
conducted. The protecting groups were removed by hydrogenation
(25-35 psi H.sub.2) in the presence of 10% palladium on carbon and
hydrochloric acid. Yields for the deprotection step ranged from
70-90%. After filtration and drying, the hydrochloride salts of the
serine and threonine esters of (.+-.)-Ibuprofen were purified by
crystallization. The hydrochloride salt of the
L-hydroxyproline-Ibuprofen ester was a gel that would not
solidify/crystallize. In this case the hydrogenation was repeated
without the use of acid and the neutral compound was purified.
[0284] Because the Ibuprofen started as a mixture of enantiomers,
the final products were delivered as a mixture of diastereomers
except for the threonine ester. In the case of the threonine ester
of Ibuprofen, washing with water, acetone or acetonitrile could
readily separate the final diastereomeric salts. The insoluble
isomer (SPI0016A) was determined to be the active isomer by
comparison with an authentic standard prepared from
S-(+)-Ibuprofen. The serine and hydroxyproline esters of
(.+-.)-Ibuprofen could not be readily separated in this fashion.
Synthetic Sequence: ##STR31## Synthesis of the L-serine,
L-threonine, and L-hydroxyproline Esters of (.+-.)-Ibuprofen: a)
EDC, DMAP, CH.sub.2Cl.sub.2; b) HCl, 10% Pd/C, EtOH c) Acetone, d)
10% Pd/C, EtOH. Experimental Section:
[0285] The synthesis of SPI0015, SPI0016 and SPI0017 were conducted
in two or three batches. Reagents mentioned in the experimental
section were purchased at the highest obtainable purity from
Sigma-Aldrich, Acros, or Bachem, except for solvents, which were
purchased from either Fisher Scientific or Mallinkrodt.
1) Preparation of (.+-.)-Ibuprofen-L-serine Ester, Hydrochloride
(SPI0015).
[0286] (.+-.)-Ibuprofen (5.04 g, 24.4 mmole),
N-carbobenzyloxy-L-serine benzyl ester (8.11 g, 24.6 mmole),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,
4.87 g, 25.4 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.40
g, 3.27 mmole) were dissolved in dichloromethane (150 mL) at room
temperature, under an argon atmosphere. After stirring for 22 hours
under an argon atmosphere at room temperature, water (100 mL) was
added and the layers were separated. The dichloromethane layer was
washed again with water (100 mL) and dried for 1 hour over sodium
sulfate (5 g). After filtration and concentration under reduced
pressure, the remaining oil was purified by flash chromatography on
silica gel (250 g), eluting with hexanes/ethyl acetate (3:1). The
procedure generated the protected L-serine-(.+-.)-Ibuprofen ester
(SPI001501) as a colorless solid (11.4 g, 90% yield). ##STR32##
2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-pr-
opionic acid benzyl ester
[0287] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.40-7.20 (m,
10H), 7.14-7.01 (m, 4H), 5.50 (d, 1/2H, J=8.4 Hz), 5.29 (d, 1/2H,
J=8.4 Hz), 5.11-5.02 (m, 2.5H), 4.90 (d, 1/2H, J=12 Hz), 4.62 (m,
1H), 4.49-4.43 (m, 1H), 4.36-4.32 (m, 1H), 3.59 (m, 1H), 2.39-2.35
(m, 2H), 1.78 (m, 1H), 1.42-1.39 (m, 3H), 0.85 (d, 6H, J=6.6
Hz).
[0288] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=174.05, 169.19,
169.07, 155.68, 140.73, 137.20, 136.12, 135.05, 134.91, 129.44,
128.67, 128.65, 128.60, 128.41, 128.33, 128.30, 128.19, 127.19,
127.16, 67.75, 67.32, 64.51, 64.32, 53.71, 45.16, 45.02, 30.35,
22.60, 18.27.
[0289] The protected Ibuprofen-L-serine ester (22.50 g, 43.4 mmole)
was dissolved in ethanol (200 mL) at room temperature and added to
a Parr bottle that contained 10% palladium on carbon (3.86 g, 50%
wet) under a nitrogen atmosphere. Hydrochloric acid (10 mL 37% HCl
in 30 mL water) was added and the nitrogen atmosphere was replaced
with hydrogen gas (25 psi). After 4 hours of shaking, the palladium
catalyst was removed by filtration through celite. The
ethanol/water was removed under reduced pressure. The remaining
white solids were washed with water (25 mL), acetone (20 mL) and
dried under high vacuum (4 hours at 88.degree. C.). The experiment
produced (.+-.)-Ibuprofen-L-serine ester, hydrochloride SPI0015
(11.3 g, 80% yield) as a colorless solid. ##STR33##
2(S)-Amino-3-[2(R,S)-(4-isobutylphenyl)-propionyloxy]-propionic
acid, hydrochloride; ((R,S)-Ibuprofen-L-Serine ester,
hydrochloride)
[0290] .sup.1H NMR (300 MHz, DMSO): .delta.=8.92 (br s, 3H), 7.22
(t, 2H, J=7.5 Hz), 7.10 (d, 2H, J=7.5 Hz), 4.56 (m, 1H), 4.37-4.20
(m, 2H), 3.83 (q, 1H, J=6.9 Hz), 2.41 (d, 2H, J=6.9 Hz), 1.80 (m,
1H), 1.41 (d, 3H, J=6.9 Hz), 0.85 (d, 6H, J=6.9 Hz).
[0291] .sup.13C NMR (75 MHz, DMSO): .delta.=173.36, 173.32, 168.08,
168.04, 139.70, 128.96, 129.92, 127.20, 127.05, 62.47, 51.59,
51.49, 44.28, 44.00, 43.90, 29.68, 22.28, 18.70, 18.42.
[0292] HPLC analysis:
[0293] 99.13% purity; rt=3.133 min; Luna C18 5 u column (sn
167917-13); 4.6.times.250 mm; 254 nm; 50% ACN/50% TFA buffer
(0.1%); 35 C; 20 ul inj.; 1 ml/min; 1 mg/mL sample size; sample
dissolved in mobile phase.
[0294] CHN analysis:
[0295] calc.: C, 58.27; H, 7.33; N, 4.25. found: C, 58.44; H, 7.46;
N, 4.25.
[0296] Melting point: 169.5-170.5.degree. C.
2a) Preparation and Separation of (.+-.)-Ibuprofen-L-threonine
Ester, Hydrochloride (SPI0016A and SPI0016B).
[0297] (.+-.)-Ibuprofen (4.15 g, 20.11 mmole),
N-carbobenzyloxy-L-threonine benzyl ester (6.90 g, 20.11 mmole),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,
3.95 g, 20.6 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.25
g, 2.0 mmole) were dissolved in dichloromethane (50 mL) at room
temperature, under an argon atmosphere. After stirring for 19
hours, the dichloromethane layer was washed with water (50 mL), 5%
hydrochloric acid (2.times.25 mL), water (25 mL), saturated sodium
bicarbonate (2.times.25 mL), and water (50 mL). After drying for
one hour over sodium sulfate (5 g), filtration, and concentration
under reduced pressure, the remaining oil was used without further
purification. The procedure generated the protected
L-threonine-(.+-.)-Ibuprofen ester (SPI001601) as a light yellow
oil (10.2 g, 95.3% yield), which solidified on standing.
##STR34##
2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-bu-
tyric acid benzyl ester
[0298] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.40-7.15 (m,
10H), 7.14-7.01 (m, 4H), 5.48-5.25 (m, 2H), 5.11-5.01 (m, 3H), 4.90
(d, 1/2H, J=12 Hz), 4.68 (d, 1/2H, J=12 Hz), 4.48 (m, 1H),
3.60-3.48 (m, 1H), 2.39(m, 2H), 1.79 (m, 1H), 1.42-1.35 (m, 3H),
1.27 (d, 1.5H, J=6.6 Hz), 1.17 (d, 1.5H, J=6.6 Hz), 0.85 (m,
6H).
[0299] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=173.32, 169.70,
169.30, 156.55, 140.75, 137.38, 137.22, 136.14, 135.07, 134.99,
129.45, 129.41, 128.65, 128.39, 128.22, 127.21, 127.14, 70.97,
70.70, 67.81, 67.66, 67.53, 57.83, 45.19, 30.39, 22.61, 18.57,
18.30, 17.18, 16.87.
[0300] The protected Ibuprofen-L-threonine ester (10.15 g, 19.0
mmole) was dissolved in warm ethanol (150 mL) and added to a Parr
bottle that contained 10% palladium on carbon (3.4 g, 50% wet)
under a nitrogen atmosphere. Hydrochloric acid (6 mL 37% HCl in 20
mL water) was added and the nitrogen atmosphere was replaced with
hydrogen gas (30 psi). After 3 hours of shaking, the palladium
catalyst was removed by filtration through celite (30 g). The
ethanol/water was removed under reduced pressure. The experiment
produced (.+-.)-Ibuprofen-L-threonine ester, hydrochloride
(SPI0016A and SPI0016B, 6.4 g, 97% crude yield) as a colorless
solid. The crude mixture of diastereomers was stirred in acetone
(200 mL) for 2 hours at room temperature under an argon atmosphere.
After 2 hours the solids (2.84 g, SPI0016A) were filtered. The
filtrate (SPI0016B, 3.0 g) was concentrated under reduced
pressure.
1.) Purification of SPI0016A (Active Isomer):
[0301] After 3 batches of the S-Ibuprofen-L-threonine ester
(SPI0016A) had been completed, the batches were combined (8.78 g
total) and crystallized three times from DIUF ("deionized
ultrafiltered") water (100 mL). Each time a small amount of
zwitterion was generated. In order to regenerate the salt, the
solid generated (from each crystallization) was dissolved in 1%
hydrochloric acid in ethanol (3 mL 37% hydrochloric acid in 100 mL
ethanol). The ethanol solution was then concentrated under reduced
pressure at room temperature. After the third crystallization and
regeneration procedure, the salt (5.6 g) was stirred in
acetonitrile (100 mL) for 44 hours at room temperature, under an
argon atmosphere. The salt was then filtered and dried under high
vacuum at 50-55.degree. until the weight was constant (5.5 g).
##STR35##
2(S)-Amino-3(R)-[2(S)-(4-isobutyl-phenyl)-propionyloxy]-butyric
acid; (S-Ibuprofen-L-threonine ester, hydrochloride, active
isomer)
[0302] .sup.1H NMR (300 MHz, DMSO): .delta.=8.76 (br s, 3H), 7.19
(d, 2H, J=8.1 Hz), 7.11 (d, 2H, J=8.1 Hz), 5.28 (dq, 1H, J=6.3, 3.6
Hz), 4.14 (q, 1H, J=3.6 Hz), 3.80 (q, 1H, J=7.2 Hz), 2.41 (d, 2H,
J=7.2 Hz), 1.80 (m, 1H), 1.37 (d, 3H, J=7.2 Hz), 1.21 (d, 3H, J=6.3
Hz), 0.85 (d, 6H, J=6.6 Hz).
[0303] .sup.13C NMR (75 MHz, DMSO): .delta.=172.66, 168.24, 139.68,
137.24, 128.95, 126.97, 67.98, 55.35, 44.23, 43.83, 29.66, 22.24,
18.52, 16.47.
[0304] CHN analysis:
[0305] calc.: C, 59.38; H, 7.62; N, 4.07. found: C, 59.17; H, 7.63;
N, 4.04.
[0306] HPLC analysis:
[0307] 98.28% purity; r.t.=6.951 min.; 60% TFA (0.1%)/40%
acetonitrile; 1 mL/min; 37.5 C; Luna C18, 3 u column (SN
167917-13), 4.6.times.250 mm; 22 ul injection.
[0308] Optical rotation: +24.5.degree.
[0309] Melting Point: 189-190.degree. C.
2) Purification of SPI0016B (Inactive Isomer):
[0310] After 3 batches of the R-Ibuprofen-L-threonine ester
(SPI0016B) had been completed, the batches were combined (9.02 g
total) and crystallized from DIUF (deionized ultra filtered) water
(50 mL). A small amount of zwitterion was generated during the
crystallization. In order to regenerate the salt, the solid
generated was dissolved in 1% hydrochloric acid in ethanol (3 mL
37% hydrochloric acid in 100 mL ethanol). The ethanol solution was
then concentrated under reduced pressure at room temperature. The
remaining salt (5.93 g) was crystallized three times from hot
toluene (100 mL) with the addition of a small amount on acetone (1
mL). The salt was then filtered and dried under high vacuum at room
temperature until the weight was constant (5.1 g). ##STR36##
2(S)-Amino-3(R)-[2(R)-(4-isobutyl-phenyl)-propionyloxy]-butyric
acid; (R-Ibuprofen-L-threonine ester, hydrochloride, inactive
isomer)
[0311] .sup.1H NMR (300 MHz, DMSO): .delta.=8.82 (br s, 3H), 7.23
(d, 2H, J=7.8 Hz), 7.10 (d, 2H, J=7.8 Hz), 5.27 (m, 1H), 4.18 (m,
1H), 3.80 (q, 1H, J=7.2 Hz), 2.41 (d, 2H, J=7.2 Hz), 1.81 (m, 1H),
1.41 (d, 3H, J=6.9 Hz), 1.34 (d, 3H, J=6.3 Hz), 0.85 (d, 6H, J=6.3
Hz).
[0312] .sup.13C NMR (75 MHz, DMSO): .delta.=72.56, 168.08, 139.64,
136.98, 128.84, 127.14, 68.8, 55.29, 44.28, 29.69, 22.28, 18.24,
16.41.
[0313] CHN analysis:
[0314] calc.: C, 59.38; H, 7.62; N, 4.07. found: C, 59.30; H, 7.60;
N, 4.05.
[0315] HPLC analysis:
[0316] 98.43% purity; r.t.=6.19 min.; 60% TFA (0.1%)/40%
acetonitrile; 1 mL/min; 37.5 C; Luna C18, 3 u column (SN
167917-13), 4.6.times.250 mm; 22 ul injection.
[0317] Optical Rotation: +10.4
[0318] Melting Point: 176-177.degree. C.
2b) Preparation of the S-(+)-Ibuprofen-L-threonine Ester,
hydrochloride Standard (SPI0016S).
[0319] S-(+)-Ibuprofen (2.0 g, 9.69 mmole),
N-carbobenzyloxy-L-threonine benzyl ester (3.25 g, 9.91 mmole),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,
1.90 g, 9.91 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.12
g, 1.0 mmole) were dissolved in dichloromethane (25 mL) at room
temperature, under an argon atmosphere. After stirring for 4 hours,
the dichloromethane layer was washed with water (25 mL), 5%
hydrochloric acid (25 mL), saturated sodium bicarbonate (2.times.25
mL), and water (25 mL). After drying for one hour over sodium
sulfate (5 g), filtration, and concentration under reduced
pressure, the remaining oil was used without further purification.
The procedure generated the protected S-(+)-Ibuprofen-L-threonine
ester (SPI001601S) as a light yellow oil (5.01 g, 98% yield), which
solidified on standing. ##STR37##
2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-bu-
tyric acid benzyl ester
[0320] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.35-7.23 (m,
10H), 7.10 (d, 2H, J=7.8 Hz), 7.05 (d, 2H, J=7.8 Hz), 5.48-5.25 (m,
2H), 5.17-5.01 (m, 4H), 4.50 (dd, 1H, J=9.6, 1.8 Hz), 3.50 (q, 1H,
J=7.2 Hz), 2.40 (d, 2H, J=7.2 Hz), 1.80 (m, 1H), 1.37 (d, 3H, J=7.2
Hz), 1.17 (d, 3H, J=6.3 Hz), 0.86 (d, 6H, J=6.6 Hz).
[0321] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=173.29, 169.69,
156.51, 140.68, 137.21, 136.08, 135.06, 129.40, 128.70, 128.66,
128.57, 128.38, 128.24, 127.14, 70.70, 67.80, 67.53, 57.87, 45.19,
45.11, 30.39, 22.61, 18.57, 16.87.
[0322] The protected S-(+)-Ibuprofen-L-threonine ester (5.0 g, 9.40
mmole) was dissolved in warm ethanol (100 mL) and added to a Parr
bottle that contained 10% palladium on carbon (1.0 g, 50% wet)
under a nitrogen atmosphere. Hydrochloric acid (1 mL 37% HCl in 10
mL water) was added and the nitrogen atmosphere was replaced with
hydrogen gas (32 psi). After 2 hours of shaking, the palladium
catalyst was removed by filtration through celite (30 g). The
ethanol/water was removed under reduced pressure. The experiment
produced S-(+)-Ibuprofen-L-threonine ester, hydrochloride
(SPI0016S, 2.8 g, 85% crude yield) as a colorless solid. The salt
was stirred in acetone (50 mL) for 3 hours at room temperature
under an argon atmosphere. After 3 hours the solids (2.24 g, 69%
yield) were filtered and dried under high vacuum at room
temperature, until the weight was constant. ##STR38##
2(S)-Amino-3(R)-[2(S)-(4-isobutyl-phenyl)-propionyloxy]-butyric
acid; (S-Ibuprofen-L-threonine ester, hydrochloride, active
isomer)
[0323] .sup.1H NMR (300 MHz, DMSO): .delta.=8.76 (br s, 3H), 7.19
(d, 2H, J=8.1 Hz), 7.11 (d, 2H, J=8.1 Hz), 5.28 (dq, 1H, J=6.3, 3.6
Hz), 4.14 (q, 1H, J=3.6 Hz), 3.80 (q, 1H, J=7.2 Hz), 2.41 (d, 2H,
J=7.2 Hz), 1.80 (m, 1H), 1.37 (d, 3H, J=7.2 Hz), 1.21 (d, 3H, J=6.3
Hz), 0.85 (d, 6H, J=6.6 Hz).
[0324] .sup.13C NMR (75 MHz, DMSO): .delta.=172.66, 168.24, 139.68,
137.24, 128.95, 126.97, 67.98, 55.35, 44.23, 43.83, 29.66, 22.24,
18.52, 16.47.
[0325] HPLC analysis:
[0326] 98.28% purity; r.t.=6.951 min.; 60% TFA (0.1%)/40%
acetonitrile; 1 mL/min; 37.5 C; Luna C18, 3 u column (SN
167917-13), 4.6.times.250 mm; 22 ul injection.
[0327] Optical rotation: +26.5.degree.
[0328] Melting Point: 189-190.degree. C.
3) Preparation of the (.+-.)-Ibuprofen-L-hydroxyproline Ester
(SPI0017).
[0329] (.+-.)-Ibuprofen (5.10 g, 24.7 mmole),
N-carbobenzyloxy-L-hydroxyproline benzyl ester (8.80 g, 24.7
mmole), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,
hydrochloride (EDC, 5.10 g, 26.0 mmole), and
4-(N,N-dimethylamino)-pyridine (DMAP, 0.30 g, 2.40 mmole) were
dissolved in dichloromethane (100 mL) at room temperature, under an
argon atmosphere. After stirring for 24 hours under an argon
atmosphere at room temperature, water (100 mL) was added and the
layers were separated. The dichloromethane layer was washed again
with water (100 mL), 5% sodium bicarbonate (2.times.50 mL) and
dried for 1 hour over sodium sulfate (5 g). After filtration and
concentration under reduced pressure, the remaining oil was used
without further purification. The procedure generated the protected
(.+-.)-Ibuprofen-L-hydroxyproline ester (SPI001701) as a light
yellow oil (11.5 g, 85% yield). ##STR39##
4(R)-[2-(4-Isobutyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylic
acid; ((R,S)-Ibuprofen-L-hydroxyproline ester)
[0330] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.33-7.02 (m,
14H), 5.25-4.95 (m, 5H), 4.51-4.19 (m, 1H), 3.75-3.50 (m, 3H), 2.40
(d, 2H, J=6.9 Hz), 2.15 (m, 1H), 1.81 (m, 1H), 1.44 (d, 3H, J=7.0
Hz), 0.87 (d, 6H, J=6.6 Hz).
[0331] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=173.99, 171.93,
171.72, 154.68, 154.15, 140.70, 137.23, 137.04, 136.23, 135.44,
135.23, 129.41, 128.59, 128.47, 128.35, 128.19, 128.08, 127.89,
127.02, 72.86, 72.16, 67.40, 67.18, 67.09, 58.12, 57.83, 52.66,
52.49, 52.13, 45.15, 36.63, 35.67, 32.07, 30.33, 29.23, 22.90,
22.58, 18.36.
[0332] The protected Ibuprofen-L-hydroxyproline ester (11.40 g,
43.4 mmole) was dissolved in ethanol (150 mL) at room temperature
and added to a Parr bottle that contained 10% palladium on carbon
(2.73 g, 50% wet) under a nitrogen atmosphere. The nitrogen
atmosphere was replaced with hydrogen gas (34 psi). After 5 hours
of shaking, the palladium catalyst was removed by filtration
through celite. The ethanol was removed under reduced pressure. The
remaining white solids (6.60 g) were washed with DIUF water (50
mL), diethyl ether (50 mL) and dried under high vacuum until the
weight was constant. The experiment produced
(.+-.)-Ibuprofen-L-hydroxyproline ester SPI0017 (5.64 g, 84% yield)
as a colorless solid. ##STR40##
4(R)-[2-(4-Isobutyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylic
acid; ((R,S)-Ibuprofen-L-hydroxyproline ester)
[0333] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.22 (d, 2H,
J=7.2 Hz), 7.09 (d, 2H, J=7.2 Hz), 5.27 (m, 1H), 4.40 (t, 0.5H, J=7
Hz), 4.24 (t, 0.5H, J=9 Hz), 3.75 (m, 1H), 3.61(m, 1H), 3.28 (d,
0.5H, J=13 Hz), 3.15 (d, 0.5H, J=13 Hz), 2.42-2.10 (m, 4H), 1.78
(m, 1H), 1.40 (br t, 3H, J=6 Hz), 0.82 (d, 6H, J=6 Hz). (mixture of
diastereomers)
[0334] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=173.28, 173.23,
168.98, 139.88, 137.33, 137.23, 129.12, 127.26, 127.17, 72.58,
57.60, 57.50, 50.24, 50.12, 44.34, 44.15, 34.31, 34.16, 29.77,
22.34, 18.43, 18.23. (mixture of diastereomers)
[0335] HPLC analysis:
[0336] 100% purity; r.t.=5.35, 5.22 min.; 55% TFA (0.1%), 45% ACN;
1 mL/min; 32.3 C, Luna C18, serial # 188255-37; 20 ul inj.
[0337] CHN analysis:
[0338] calc.: C, 67.69; H, 7.89; N, 4.39. found: C, 67.47; H, 7.87;
N, 4.30.
[0339] Melting Point: 198-199.degree. C.
Efficacy (Anti Nociceptive Potential) of Synthesis of the L-serine,
L-threonine, and L-hydroxyproline Esters of (.+-.)-Ibuprofen by
Employing Acetylcholine Induced Abdominal Constriction Method in
Male Albino Mice:
[0340] The present study was conducted to evaluate the efficacy of
L-serine, L-threonine, and L-hydroxyproline esters of
(.+-.)-Ibuprofen taking into account the antagonizing property on
acetylcholine induced writhe as an index in albino mice. Ibuprofen
(racemic mixture) and ibuprofen (S)-(+) served as reference
controls.
[0341] Different new formulations of ibuprofen and reference
controls viz., ibuprofen (racemic mixture) and ibuprofen (S)-(+)
were administered by gavage to male albino mice (Swiss strain),
using 5% (v/v) Tween 80 in milli Q water as the vehicle. The study
was conducted at two dose levels viz. 50 mg and 100 mg/kg body
weight along with a vehicle control group. At each dose level 10
animals were used. All the doses were expressed as ibuprofen molar
equivalents. The doses used as well as the molar equivalents are
presented below. TABLE-US-00006 TABLE 5 Formulation: Molar
Equivalent: Formulation Molar equivalent S-(+)-Ibuprofen-L- 0.833
units are equivalent to 1 unit of Ibuprofen threonine ester
(.+-.)-Ibuprofen-L- 1.6 units are equivalent to 1 unit of Ibuprofen
serine ester (.+-.)-Ibuprofen-L- 1.55 units are equivalent to 1
unit of Ibuprofen hydroxyproline ester
[0342] TABLE-US-00007 TABLE 6 Test Item: Group: Dose(mg/kg):
Equivalent wt. Of the test item: Equivalent Dose (mg per kg) weight
of the [in terms of Test item Test Item Group Ibuprofen] [mg/kg]
Vehicle Vehicle control 0.0 -- Group S-(+)-Ibuprofen-L- Test Group
1 50.0 41.65 threonine ester Test Group 2 100.0 83.30
(.+-.)-Ibuprofen-L- Test Group 3 50.0 80.0 serine ester Test Group
4 100.0 160.0 (Ibuprofen S) (.+-.)-Ibuprofen-L- Test Group 5 50.0
77.5 hydroxyproline ester Test Group 6 100.0 155.0 Ibuprofen
(racemic Test Group 7 50.0 50.0 mixture) Test Group 8 100.0 100.0
Ibuprofen S+ Test Group 9 50.0 25.0 Test Group 10 100.0 50.0
[0343] The efficacy in terms of antagonizing effect on
acetylcholine induced single writhe at two dose levels--50.0 and
100.0 mg/kg for the three formulations and reference controls are
presented below. TABLE-US-00008 TABLE 7 Test Item: Group: Dose
(mg/kg): Number of animals showing absence of single writhe (out of
10) Number of animals showing absence of single writhe (number of
animals Dose (mg per dose = 10) per kg) One hour [in terms of after
Three hours Test Item Group Ibuprofen] dosing after dosing Vehicle
Vehicle control 0.0 0 0 S-(+)- Low dose 50.0 1 0 Ibuprofen-L- High
dose 100.0 3 0 threonine ester (.+-.)- Low dose 50.0 4 2
Ibuprofen-L- High dose 100.0 6 4 serine ester (.+-.)- Low dose 50.0
5 4 Ibuprofen-L- High dose 100.0 7 7 hydroxyproline ester Ibuprofen
Low dose 50.0 4 2 (racemic High dose 100.0 6 6 mixture) Ibuprofen
S+ Low dose 50.0 5 1 High dose 100.0 6 6
[0344] Statistical analysis employing Chi--square test procedure
did not show any statistically significant difference among the
formulations in comparison to reference control, while comparing
the number of animals not showing writhe in each groups, as the
respective "p" was found to be greater than 0.05, the level of
significance.
[0345] From clinical observation based on the number of animals not
showing writhes due to administration of acetylcholine,
(.+-.)-Ibuprofen-L-hydroxyproline ester was found to be more
effective in antagonizing the acetylcholine induced writhe when
compared to other formulations and Ibuprofen (racemic) and
Ibuprofen (S)-(+). TABLE-US-00009 TABLE 8 Summary of Efficacy of
L-serine, L-threonine, and L-hydroxyproline esters of
(.+-.)-Ibuprofen, Ibuprofen (racemic mixture) and Ibuprofen (S)-(+)
- Based on Antagonizing Property of Acetylcholine Induced Writhe in
Albino Mice Number of animals showing absence of Dose single writhe
(number (mg/kg) of animals per dose = 10) [in terms of One hour
after Three hours Ibuprofen] Test Item dosing after dosing 50 mg/kg
Vehicle control 0 0 S-(+)-Ibuprofen-L- 1 0 threonine ester
(.+-.)-Ibuprofen-L- 4 2 serine ester (.+-.)-Ibuprofen-L- 5 4
hydroxyproline ester Ibuprofen 4 2 (racemic mixture) Ibuprofen
(S)-(+) 5 1
[0346] TABLE-US-00010 TABLE 9 100 mg/kg Vehicle control 0 0
S-(+)-Ibuprofen- 3 0 L-threonine ester (.+-.)-Ibuprofen-L- 6 4
serine ester (.+-.)-Ibuprofen-L- 7 7 hydroxyproline ester Ibuprofen
6 6 (racemic mixture) Ibuprofen (S)-(+) 6 6
[0347] The data were subjected to statistical analysis employing
Chi--square test procedure for evaluating the efficacy of the new
formulations in comparison to the reference controls. The tests did
not show any statistically significant difference among the
formulations in comparison to reference control, while comparing
the number of animals not showing writhe in each groups, as the
respective "p" was found to be greater than 0.05, the level of
significance.
[0348] The data is also summarized in FIGS. 1 and 2. From clinical
observations and bar diagram for comparative efficacy (FIGS. 1 and
2), based on the number of animals not showing writhes due to
administration of acetylcholine, (.+-.)-Ibuprofen-L-hydroxyproline
ester was found to be more effective in antagonizing the
acetylcholine induced writhe when compared to other formulations
and Ibuprofen (racemic) and Ibuprofen (S)-(+).
Conclusion
[0349] The present study was conducted to evaluate the relative
efficacy of new formulations of ibuprofen. For this the
antagonizing property of new formulations on acetylcholine writhes
was taken as an index to determine the relative efficacy of the
formulations. Ibuprofen (racemic mixture and ibuprofen (S)-(+)
served as reference controls. The study was conducted at two dose
levels (50.0 and 100.0 mg/kg) along with a vehicle control
group.
[0350] The efficacy in terms of antagonizing effect of
acetylcholine induced single writhe at two dose levels--50.0 and
100.0 mg/kg for the three formulations and reference controls are
presented below. TABLE-US-00011 TABLE 10 Test Item: Group: Dose
(mg/kg): No. of animals showing absence of single writhe (out of
10) Number of animals Dose (mg showing absence of per kg) single
writhe (number [in of animals per dose = 10) terms of One hour
after Three hours Test Item Group Ibuprofen] dosing after dosing
Vehicle Vehicle 0.0 0 0 control S-(+)-Ibuprofen- Low dose 50.0 1 0
L-threonine High dose 100.0 3 0 ester (.+-.)-Ibuprofen-L- Low dose
50.0 4 2 serine ester High dose 100.0 6 4 (.+-.)-Ibuprofen-L- Low
dose 50.0 5 4 hydroxyproline High dose 100.0 7 7 ester Ibuprofen
Low dose 50.0 4 2 (racemic High dose 100.0 6 6 mixture) Ibuprofen
(S)- Low dose 50.0 5 1 (+) High dose 100.0 6 6
[0351] Statistical analysis employing Chi--square test procedure
did not show any statistically significant difference among the
formulations in comparison to reference control, while comparing
the number of animals not showing writhe in each groups, as the
respective "p" was found to be greater than 0.05, the level of
significance.
[0352] However from clinical observation based on the number of
animals not showing writhes due to administration of acetylcholine
(.+-.)-Ibuprofen-L-hydroxyproline ester was found to be more
effective in antagonizing the acetylcholine induced writhe when
compared to other formulations and Ibuprofen (racemic) and
Ibuprofen (S)-(+).
Gastric Mucosal Irritation Potential of L-serine, L-threonine, and
L-hydroxyproline Esters of (.+-.)-Ibuprofen in Fasted Male Albino
Rats
Summary
[0353] The present study was conducted to determine the relative
potential of new formulations of ibuprofen (L-serine, L-threonine,
and L-hydroxyproline esters of (.+-.)-Ibuprofen) to cause gastric
mucosal irritation/lesions in fasted male albino rats. Ibuprofen
(racemic mixture) and Ibuprofen(S)-(.+-.) served as reference
controls.
[0354] Different new formulations of ibuprofen and ibuprofen
(racemic mixture) and ibuprofen(S)-(+) were administered by gavage
to fasted male albino rats (Wistar strain), using 5% solution of
Tween 80 in milli Q water as the vehicle. The study was conducted
at two dose levels viz. 200 mg and 300 mg/kg body weight along with
a vehicle control group. At each dose level 5 animals were used.
All the doses were expressed as ibuprofen (racemic mixture) molar
equivalents. The doses used as well as the molar equivalents were
presented below. TABLE-US-00012 TABLE 11 Formulation: Molar
Equivalent Formulation Molar equivalent S-(+)-Ibuprofen-L- 0.833
units are equivalent to 1 unit of Ibuprofen threonine ester
(.+-.)-Ibuprofen-L- 1.60 units are equivalent to 1 unit of
Ibuprofen serine ester (.+-.)-Ibuprofen-L- 1.55 units are
equivalent to 1 unit of Ibuprofen hydroxyproline ester
[0355] The various groups used are tabulated hereinbelow:
TABLE-US-00013 TABLE 12 Test item: group: Dose (mg/kg) Equivalent
wt. Equivalent weight of Dose (mg per kg) the [in terms of Test
item Test item Group Ibuprofen] [mg/kg] Vehicle Vehicle control 0.0
-- Group S-(+)-Ibuprofen-L- Test Group 1 200.0 0.0 threonine ester
Test Group 2 300.0 166.6 (.+-.)-Ibuprofen-L-serine Test Group 1
200.0 249.9 ester Test Group 2 300.0 320.0 (.+-.)-Ibuprofen-L- Test
Group 1 200.0 480.0 hydroxyproline ester Test Group 2 300.0 310.0
Ibuprofen (racemic Test Group 1 200.0 465.0 mixture) Test Group 2
300.0 300.0 Ibuprofen (S)-(+) Test Group 1 200.0 100.0 Test Group 2
300.0 150.0
[0356] The rats were fasted for a period of 18 to 22 hours before
dosing. The test item was administered as a single dose by gavage.
Three hours after drug administration, the animals were killed
humanely by CO.sub.2 gas inhalation. The stomach was dissected out
and observed for [0357] the quantity of mucous exudate, [0358]
degree of hyperemia and thickening of stomach wall, [0359]
hemorrhagic spots (focal or diffuse), nature of hemorrhages
(petechial or ecchymotic) along with the size and [0360]
perforations or any other lesions
[0361] The observations on gastric mucosal irritation of animals of
various groups were summarized below: TABLE-US-00014 TABLE 13 Test
item: Group: Dose (mg/kg): Observation Doe mg/kg (as per Test Item
Group ibuprofen) Observation Vehicle control Vehicle control 0.0
None of the animals Group showed any evidence of gastric mucosal
irritation S-(+)-Ibuprofen- Test Group 1 200.0 None of the dosed
L-threonine ester animals showed any evidence of gastric mucosal
irritation Test Group 2 300.0 None of the dosed animals showed any
evidence of gastric mucosal irritation. (.+-.)-Ibuprofen-L- Test
Group 1 200.0 None of the dosed serine ester animals showed any
evidence of gastric mucosal irritation. Test Group 2 300.0 None of
the dosed animals showed any evidence of gastric mucosal irritation
(.+-.)-Ibuprofen-L- Test Group 1 200.0 None of the dosed
hydroxyproline animals showed any ester evidence of gastric mucosal
irritation Test Group 2 300.0 None of the dosed animals showed any
evidence of gastric mucosal irritation Ibuprofen Test Group 1 200.0
Gastric mucosal (racemic mixture) irritation was observed in one
animal out of 5 animals dosed. Test Group 2 300.0 Gastric mucosal
irritation was observed in two animals out of 5 animals dosed.
Ibuprofen (S)- Test Group 1 200.0 Gastric mucosal (+) irritation
was observed in all the 5 animals dosed. Test Group 2 300.0 Gastric
mucosal irritation was observed in three animals out of 5 animals
dosed.
[0362] The results of the present study showed that none of the
formulations of ibuprofen had caused any evidence of irritation of
gastric mucosa in fasted male albino rats of male sex at the two
dose levels tested (200 mg and 300 mg/kg body weight). In contrast,
both ibuprofen (racemic mixture) and ibuprofen (S)-(+) had caused
irritation of gastric mucosa at the two dose levels tested. Further
ibuprofen(S)-(+) was found to be more gastric mucosal irritant than
ibuprofen (racemic mixture).
28-Day Chronic Toxicity Studies with S(+) Ibuprofen L-Threonin
Ester in Rats
[0363] Chronic toxicity of S(+) Ibuprofen-L-Threonine ester was
compared against a vehicle, (+/-)racemic Ibuprofen and (+/-)racemic
Ibuprofen-L-Hydroproline ester. Test species used was Swiss Albino
Mice, both male and female with body weight range of 18-27 gms.
Randomization was done by the method of stratified randomization
procedure using SAS software program (Version 8.2) with
stratification by bodyweight. TABLE-US-00015 TABLE 4 NUMBER OF
ANIMAL NUMBERS GROUP TEST ITEM ANIMALS Female Male Vehicle Control
Vehicle 10 01 to 05 06 to 10 Test Group 1 Formulation 1 10 11 to 15
16 to 20 of Ibuprofen Test Group 2 Formulation 5 10 21 to 25 26 to
30 of Ibuprofen Reference Test Ibuprofen USP 10 31 to 35 36 to 40
Group
[0364] The test doses are expressed as Ibuprofen molar equivalents:
TABLE-US-00016 TABLE 5 Dose (mg per kg) Equivalent [in terms of
weight of the Test Item Group Ibuprofen] Test item [mg] Vehicle
Vehicle 0.0 0.0 Control L-Threonine ester of Test Group 1 200.0
334.0 S(+) Ibuprofen L-Hydroxyproline ester Test Group 2 200.0
310.0 of Racemin Ibuprofen Ibuprofen USP Reference 200.0 200.0
(Racemic mixture) Control
[0365] The duration of dosing was 28 days. All the animals were
daily tested until the end of the study for the presence/absence of
clinical symptoms of toxicity. Cage side observations included
changes in the skin, eyes, posture, gait, respiration and behavior
pattern. The incidence of twitching, tremors, convulsions,
salivation, diarrhea and death if any, were also recorded.
[0366] Animals Exposed to Different Doses of the Test Substance Did
not Indicate any Symptoms of Toxicity (Table 16). TABLE-US-00017
TABLE 16 Summary of Clinical Symptoms of Toxicity in Albino Mice
Period of GROUP Animal Signs in days (mg/kg body weight) Symptoms
of toxicity Sex Numbers From-to Mortality Vehicle Control (0.0) No
symptoms of Female 01 to 05 0-28 Nil (No Treatment) toxicity were
observed No symptoms of Male 6 to 10 0-28 Nil toxicity were
observed Test Group 1 No symptoms of Female 11 to 15 0-28 Nil
(Ibuprofen S + T) toxicity were observed (334.0 mg/kg) No symptoms
of Male 16 to 20 0-28 Nil toxicity were observed Test Group 2 No
symptoms of Female 21 to 25 0-28 1/5 (Ibuprofen HP) toxicity were
observed (310.0 mg/kg) No symptoms of Male 26 to 30 0-28 Nil
toxicity were observed Reference Control 3 No symptoms of Female 31
to 35 0-28 3/5 (Ibuprofen USP) toxicity were observed (200.0 mg/kg)
No symptoms of Male 36 to 40 0-28 1/5 toxicity were observed In the
above table, S + T Ibuprofen refers to S(+)Ibuprofen-L-Threonine
Ester.
Death Record
[0367] Ibuprofen HP
[0368] Animal no. 23 (female animal) died on 21 day of dosing.
[0369] Positive Control Group
[0370] Animal no. 31 (female animal) died on 23 day of dosing.
[0371] Animal no. 32 (female animal) died on 21 day of dosing.
[0372] Animal no. 33 (female animal) died on 24 day of dosing.
[0373] Animal no. 40 (male animal) died on 10 day of dosing.
[0374] While there were no cage side specific toxicity were noted,
surprisingly 40% of the animals receiving racemic ibuprofen died,
only 10% of the rats receiving Hydroxyproline ester of Iburprofen
did not complete the full course, and even more surprisingly, none
of the animals in the S(+)Ibuprofen-L-Threonine ester group died.
The average increase/decrease in body weight and the percentage
change of body weight of the surviving animals in various groups
are shown below: TABLE-US-00018 TABLE 17 Average Change Percentage
Change Treatment In body Weight (gms) (No of animals survived)
Vehicle 4.65 19.97 (10) S(+)Ibuprofen -0.68 -3.61 (10) Threonine
Ester Ibuprofen Hydroxy- 1.43 5.75 (9) Proline Ester Racemic 2.97
12.54 (6) Ibuprofen
[0375] While there was increase in body weight in treatments with
racemic Ibuprofen and Ibuprofen Hydroxyproline ester, both group
have mortalities, with currently marketed Ibuprofen showing more
mortality than Hydroxyproline ester. Hence all the amino acid
esters are far superior to Ibuprofen racemic mixture or the active
S(+)Ibuprofen. However, the best product so far seems to be
S(+)Ibuprofen-L-Threonine Ester, making it one of the ideal
candidates to be advanced to human trials.
Human Clinical Trials with S(+)Ibuprofen-L-Threonine Ester:
[0376] Determination of the analgesic and anti-inflammatory effects
of S(+)Ibuprofen-L-Threonine Ester in three human volunteers was
conducted as follows:
[0377] Two male, age 49 and 50 having severe headache took 1
capsule containing S(+)Ibuprofen-L-Threonine Ester manufacturing
and formulation under GMP conditions. The capsule contents were
equivalent to 200 mg of racemic Ibuprofen. Relief from headache was
reported after 15 min, and complete absence of any pain from
headache was reported at the end of 1 hour, which lasted for
another 12 hours.
[0378] Two males age 49 and 51 took 1 capsule each containing
S(+)IbuprofenL-Threonine ester for arthritic knee pain, which was
perceptible. After 12 hours, both volunteers reported significant
reduction is the pain associated with their right knee. Such
amolearation of pain was further sustained for another 24
hours.
Pharmacokinetics of Ibuprofen in Human Volunteers:
[0379] Based upon preliminary analgesic and anti-inflammatory
response from the 4 volunteers, Ibuprofen racemic drug was compared
against S(+)Ibuprofen-L-Threonine ester at 200 mg equivalent dose.
The plasma-concentration time profile in 6 volunteers, where
S(+)Ibuprofen-L-Threonine ester concentrations were plotted against
racemic ibuprofen concentrations in plasma for each volunteers.
[0380] Based upon the results of comparative bioavailablity of
racemic Ibuprofen versus Ibuprofen released from
S(+)Ibuprofen-L-Threonine ester it is clear that only very small
amount of Ibuprofen is released intact into human blood stream.
This is due to the fact that S(+)Ibuprofen-L-threonine ester does
not act as a derivative of Ibuprofen, instead the Threonine ester
had intact activity.
[0381] The overall bioavailability of Ibuprofen from Ibuprofen
Racemic mixture of 200 mg and equivalent dose of
S(+)Ibuprofen-L-Threonine ester are shown in the table below:
TABLE-US-00019 TABLE 18 Volunteer AUC1 AUC2 % Availability 1
69197.618 893.226 1.3 2 41861.277 1978.925 4.7 3 73121.747 940.133
1.3 4 38993.502 2101.642 5.4 5 34567.246 1657.496 4.8 6 66710.152
925.000 1.4
[0382] As used herein, the term AUC refers to area under the curve.
In the above table, AUC1 represents the cumulative area under the
plasma concentration time curve following oral administration of
200 mg of racemic ibuprofen to human volunteers, and AUC2
represents the cumulative area under the plasma concentration time
curve following oral administration of 200 mg ibuprofen equivalence
of S(+)Ibuprofen-L-Threonine ester. The third column in the above
table shows relative bioavailability of Ibuprofen in human plasma
after oral administration of S(+)Ibuprofen-L-Threonine ester at
equivalent doses. This clearly demonstrates that any activity seen
in human volunteers is not due to release of any significant
amounts of S(+)Ibuprofen in to the plasma after oral ingestion of
the Threonine ester.
[0383] The reason that S(+)Ibuprofen advanced to human
pharmacokinetic studies was due to surprising results of lack of
toxicity in 28-day chronic administration in rats compared to
Ibuprofen, or Hydroxyproline ester of Ibuprofen. Furthermore,
earlier studies indicated that S(+)Ibuprofen is highly toxic to
gastric mucosa of rats (see table X above). Similar results were
also shown in various studies elsewhere, for example, other
investigators compared S(+) and R(-) enantiomers of ibuprofen in
male Wistar rats. At 40 mg/kg dose, microscopic evaluation of the
GI tissue samples revealed significance difference in GI toxicity
caused by S(+) than R(-) Ibuprofen, consistent with current
inventors results (Janjikhel, R K, Bricker, J D, Borochovitz, D,
Adeyeye, C M, Stereoselective Disposition of Sustained Release
Microspheres of Ibuprofen Enantiomers in Rats: II, Acute
Gastrointestinal Toxicity. Drug Delivery, Vol 6, No. 3, August
1999, pp 163-170). However S(+)Ibuprofen-L-Threonine ester was GI
sparing, and had no toxicity.
[0384] Furthermore, other authors have claimed that R(-)Ibuprofen
may be capable of inhibiting both therapeutic and toxic effects of
S(+)Ibuprofen (Kaehler, S T, Phleps, W, Hesse, E. Dexibuprofen:
Pharmacology, therapeutic uses and safety. Infammopharmacology, Vol
11 No. 4-6, 2003, pp 371-383). This is also consistent with current
inventor's observation, since in acute GI toxicity studies racemic
ibuprofen was somewhat less toxic than S(+)Ibuprofen.
[0385] Similarly, Rainsford K D, in Pharmacology and Toxicology of
Ibuprofen. In: Rainsford, K D, ed. Ibuprofen, A critical
bibliographic Review. London, Taylor and Francis, 2000, states that
competition between the enantiomers of ibuprofen for prostaglandin
production in vitro was evident, and that inhibition of binding of
S(+) ibuprofen by R(-) ibuprofen in the racemic mixture contributed
to the GI tolerance of the racemate.
[0386] However, what is not known in the previous art is that an
amino acid derivative, e.g., an ester of S(+)Ibuprofen would also
be nontoxic. For example, one trained in the art of such derivative
pharmacology, would have concluded that as S(+)Ibuprofen is highly
toxic to the GI mucosa, and since S(+)Ibuprofen will be released
from S(+)Ibuprofen-L-Threonine ester by the esterase enzymes in GI
tract, and by the action of pancreatin enzyme in the duodenum, it
was expected that there will not be any reduction in toxicity.
However, the current inventor surprisingly noted that
S(+)Ibuprofen-L-Threonine ester is significantly and completely
non-toxic to GI mucosa in the rats tested.
[0387] In spite of no ibuprofen appearing in the human plasma after
oral administration of S(+)Ibuprofen-L-Threonine ester, significant
analgesic and anti-inflammatory response was seen in the 2
volunteers each tested twice. This is novel since it appears, that
S(+)Ibuprofen-L-threonine ester is not a derivative, and it may
have intact pharmacological activity. This is also not anticipated
from the prior art.
[0388] While less effectiveness was seen in rat model with
S(+)Ibuprofen-L-Threonine ester, the overriding factors that
demonstrate that this drug is more suitable for human treatment of
various diseases such as arthritis etc., is due to the fact that on
chronic toxicity trials none of the animal died in this drug group.
Furthermore, no toxicity either gastric or whole animal (as
determined by number of surviving rats), was observed in the rat
model for S(+)-Ibuproten L-Threonine ester. Further, there is no
toxicity potential in the systemic circulation in human subjects.
The S(+)Ibuprofen-L-Threonine ester exhibits pharmacological
actions such as analgesic, anti-inflammatory and anti-pyretic
properties when administered to humans.
Synthesis of Ketoprofen Derivatives:
Overview:
[0389] The procedure for the synthesis of the L-threonine esters of
Ketoprofen is outlined in Synthetic Sequence section. The complete
procedure and analytical data is given in the Experimental Section.
In general, (.+-.)-Ketoprofen (5 g) was coupled with
N-boc-L-threonine t-butyl ester (1 equivalent) with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,
1 equivalent) in the presence of a catalytic amount of
4-(N,N-dimethyamino)-pyridine (DMAP). Once the reaction was
complete, any excess EDC was removed by extraction with water, DMAP
was removed by extraction with dilute acid, and Ketoprofen was
removed by extraction with sodium bicarbonate. After drying over
sodium sulfate, filtration, and concentration, the crude protected
L-threonine-(.+-.)-Ketoprofen was purified by flash chromatography
on silica gel to generate the protected L-threonine ester in good
yield (98%). The protecting groups were removed by treatment with
2M hydrochloric acid in diethyl ether to cleave the boc group,
followed by treatment with trifluoroacetic acid to remove the
t-butyl ester. After drying, the mixture of
L-threonine-R,S(.+-.)-Ketoprofen esters was separated by
crystallization from acetonitrile. The hydrochloride salt of the
L-threonine-S(+)-Ketoprofen ester preferentially precipitated from
acetonitrile. A sample of an optically pure standard was prepared
starting with S(+)-ketoprofen for comparison. After drying and
analysis, a sample of L-threonine-S(+)-Ketoprofen ester,
hydrochloride (1.75 g) separated from the mixture was shipped to
Signature Pharmaceuticals, Inc. for testing.
[0390] The L-serine and L-hydroxyproline esters of
(.+-.)-Ketoprofen were also prepared in the same manner. Attempts
to separate the mixture of Ketoprofen diastereomers by
crystallization, using a variety of solvents, failed to separate
the mixture. Since only partial separation could be seen by HPLC
analysis, the esters were purified as the mixture of diastereomers
before final analysis. The preparation and final analytical data
for the serine and hydroxyproline esters are also included.
Synthetic Sequence: ##STR41## Synthesis of the L-threonine Esters
of (.+-.)-Ketoprofen: a) EDC, DMAP, CH.sub.2Cl.sub.2; b) HCl (2M);
c) TFA; d) ACN (Crystallization). Experimental Section:
[0391] The synthesis of SPI0018A was conducted in a single batch.
Reagents mentioned in the experimental section were purchased at
the highest obtainable purity from Sigma-Aldrich, Acros, or Bachem,
except for solvents, which were purchased from either Fisher
Scientific or Mallinkrodt.
Preparation and Separation of S(+)-Ketoprofen-L-threonine Ester,
Hydrochloride (SPI0018A).
[0392] (.+-.)-Ketoprofen (5.32 g, 20.92 mmol),
N-t-butylcarbonyl-L-threonine t-butyl ester (Boc-Thr-OtBu, 5.17 g,
18.72 mmol, prepared by the literature method),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,
4.0 g, 20.9 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.22
g) were dissolved in dichloromethane (50 mL) at room temperature,
under an argon atmosphere. After stirring for 5 hours, the
dichloromethane layer was washed with water (50 mL), 5%
hydrochloric acid (2.times.25 mL), water (25 mL), saturated sodium
bicarbonate (2.times.25 mL), and water (50 mL). After drying for
one hour over sodium sulfate (5 g), filtration, and concentration
under reduced pressure, the remaining oil (10.3 g) was purified by
column chromatography on silica gel (150 g), eluting with
hexanes/ethyl acetate (2:1). After combining the product containing
fractions, concentration and drying under high vacuum, the
procedure generated the protected L-threonine-(.+-.)-Ketoprofen
ester (SPI001801) as a clear oil (9.42 g, 98% yield). ##STR42##
3-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-2(S)-tert-butoxycarbonylamino-b-
utyric acid tert-butyl ester: (mix of diastereomers)
[0393] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.83-7.42 (m,
9H), 5.43 (dd, 1H, J=13.2, 6.9 Hz), 5.10 (dd, 1H, J=20.7, 9.3),
4.29 (t, 1H, J=11.7 Hz), 3.75 (q, 1H, J=7.2 Hz), 1.50-1.42 (m,
19.5H), 1.30-1.18 (m, 4.5H).
[0394] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=. 196.18, 172.62,
172.55, 168.85, 168.58, 155.81, 140.33, 140.23, 137.86, 137.39,
132.46, 132.42, 131.54, 131.38, 130.00, 129.31, 129.13, 129.02,
128.54, 128.27, 82.50, 82.37, 80.05, 71.38, 71.22, 57.59, 57.52,
45.46, 45.31, 28.40, 27.98, 27.84, 18.54, 18.48, 17.19, 16.84.
[0395] The protected (R,S)-Ketoprofen-L-threonine ester (9.42 g,
18.41 mmol) was dissolved in dichloromethane (25 mL) under an argon
atmosphere, at room temperature. Anhydrous hydrochloric acid in
diethyl ether (2M, 25 mL) was added to the solution and the mixture
was allowed to stir for 17 hours at room temperature. The mixture
was concentrated under reduced pressure. The remaining foam (8.2 g)
was dissolved in a mixture of dichloromethane (10 mL) and
trifluoroacetic acid (20 mL). After stirring at room temperature
for 6.5 hours the solution was concentrated under reduced pressure.
Toluene (25 mL) was added to the remaining oil and the mixture was
concentrated a second time. A mixture of ethanol (20 mL) and
anhydrous hydrochloric acid in diethyl ether (2M, 20 mL) was added
and the solution was concentrated a third time. After drying under
high vacuum for 2 hours at room temperature, the experiment
produced (.+-.)-Ketoprofen-L-threonine ester, hydrochloride
(mixture of diastereomers, 7.11 g, 98% crude yield) as an off-white
solid. The crude mixture of diastereomers (7.0 g) was crystallized
3 times from acetonitrile (200 mL). After the third
crystallization, the remaining white solid was dried under high
vacuum at 50.degree. C. until the weight was constant (4 hours).
The experiment produced L-threonine-S(+)-Ketoprofen ester,
hydrochloride SPI0018A (2.2 g, 30% yield from SPI001801).
##STR43##
2(S)-Amino-3(R)-[2(S)-(3-benzoyl-phenyl)-propionyloxy]-butyric
acid, hydrochloride (L-threonine-S(+)-Ketoprofen ester,
hydrochloride)
[0396] .sup.1H NMR (300 MHz, DMSO): .delta.=14.08 (br s, 1H), 8.72
(br s, 3H), 7.74-7.51 (m, 9H), 5.29 (t, 1H, J=4.5 Hz), 4.16 (m,
1H), 3.97 (q, 1H, J=6.3 Hz), 1.42 (d, 3H, J=6.9 Hz), 1.23 (d, 3H,
J=6.3 Hz).
[0397] .sup.13C NMR (75 MHz, DMSO): .delta.=195.34, 172.26, 168.21,
140.42, 137.05, 136.74, 132.66, 131.66, 129.48, 128.73, 128.49,
128.30, 68.23, 55.31, 44.00, 18.44, 16.45.
[0398] CHN analysis:
[0399] calc.: C, 61.30; H, 5.66; N, 3.57. found: C, 61.02; H, 5.58;
N, 3.58.
[0400] HPLC analysis:
[0401] 98.28% purity; r.t.=25.14 min.; 55% DIUF water (0.1%
TFA)/45% methanol; 1 mL/min; 36.4 C; Luna C18, 5 u column (serial #
211739-42), 4.6.times.250 mm; 20 ul injection.
[0402] Optical rotation: +27.0.degree. (20 C, 174.4 mg/10 mL
ethanol, 589 nm)
[0403] Melting Point: 166-167.degree. C.
Preparation of the S-(+)-Ketoprofen-L-threonine Ester,
Hydrochloride Standard.
[0404] (+)-Ketoprofen (1.87 g, 7.74 mmol),
N-t-butylcarbonyl-L-threonine t-butyl ester (Boc-Thr-OtBu, 2.25 g,
8.14 mmol, prepared by the literature method),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,
1.65 g, 8.60 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.1
g) were dissolved in dichloromethane (25 mL) at room temperature,
under an argon atmosphere. After stirring for 4 hours, the
dichloromethane layer was washed with water (25 mL). After drying
for one hour over sodium sulfate (5 g), filtration, and
concentration under reduced pressure, the remaining oil was used
without purification. The procedure generated the protected
L-threonine-(+)-Ketoprofen ester as a clear oil (4.01 g,
.about.100% yield). ##STR44##
[0405] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.81-7.42 (m,
9H), 5.43 (m, 1H), 5.10 (d, 1H, J=9.3), 4.29 (d, 1H, J=9.6 Hz),
3.75 (q, 1H, J=7.2 Hz), 1.50-1.42 (m, 21H), 1.18 (d, 3H, J=6.3
Hz).
[0406] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=196.4, 172.79,
168.99, 155.94, 140.44, 137.99, 137.51, 132.59, 131.50, 130.13,
129.31, 129.25, 129.15, 128.66, 128.40, 82.68, 80.24, 71.37, 57.71,
45.43, 28.53, 28.10, 18.99, 16.96.
[0407] The protected (S)-Ketoprofen-L-threonine ester (3.92 g, 7.66
mmol) was dissolved in anhydrous hydrochloric acid in diethyl ether
(2M, 50 mL) and stirred for 17 hours at room temperature. The
mixture was concentrated under reduced pressure. The remaining foam
(3.4 g) was dissolved in a mixture of dichloromethane (20 mL) and
trifluoroacetic acid (20 mL). After stirring at room temperature
for 6.5 hours the solution was concentrated under reduced pressure.
Toluene (25 mL) was added to the remaining oil and the mixture was
concentrated a second time. A mixture of ethanol (20 mL) and
anhydrous hydrochloric acid in diethyl ether (2M, 20 mL) was added,
and the solution was concentrated a third time. After drying under
high vacuum for 2 hours at room temperature, the experiment
produced S(+)-Ketoprofen-L-threonine ester, hydrochloride (3.05 g
crude) as an off-white solid. The crude material was stirred with
acetone (50 mL) for 2 hours at room temperature under an argon
atmosphere. The remaining white solid was filtered and dried under
high vacuum at 50.degree. C. until the weight was constant (4
hours). The experiment produced L-threonine-S(+)-Ketoprofen ester,
hydrochloride (2.04 g, 67% yield).
[0408] .sup.1H NMR (300 MHz, DMSO): .delta.=14.08 (br s, 1H), 8.72
(br s, 3H), 7.74-7.51 (m, 9H), 5.29 (t, 1H, J=4.5 Hz), 4.16 (m,
1H), 3.97 (q, 1H, J=6.3 Hz), 1.42 (d, 3H, J=6.9 Hz), 1.23 (d, 3H,
J=6.3 Hz).
[0409] .sup.13C NMR (75 MHz, DMSO): .delta.=195.34, 172.26, 168.21,
140.42, 137.05, 136.74, 132.66, 131.66, 129.48, 128.73, 128.49,
128.30, 68.23, 55.31, 44.00, 18.44, 16.45.
[0410] HPLC analysis:
[0411] 99.43% purity; r.t.=25.14 min.; 55% DIUF water (0.1%
TFA)/45% methanol; 1 mL/min; 36.4 C; Luna C18, 5 u column (serial #
211739-42), 4.6.times.250 mm; 20 ul injection.
[0412] Optical rotation: +27.1.degree. (20 C, 177.8 mg/10 mL
ethanol, 589 nm)
[0413] Melting Point: 166-167.degree. C.
Preparation of the (.+-.)Ketoprofen-L-serine Ester,
Hydrochloride
[0414] (.+-.)-Ketoprofen (7.30 g, 28.7 mmol),
N-t-butylcarbonyl-L-serine t-butyl ester (Boc-Ser-OtBu, 7.50 g,
28.7 mmol, prepared by the literature method),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,
5.5 g, 28.7 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.12
g) were dissolved in dichloromethane (50 mL) at room temperature,
under an argon atmosphere. After stirring for 3 hours, the
dichloromethane layer was washed first with water (50 mL), then 5%
hydrochloric acid (2.times.25 mL), then water (25 mL) again,
saturated sodium bicarbonate (2.times.25 mL), and water (50 mL) a
third time. After drying for one hour over sodium sulfate (5 g),
filtration, and concentration under reduced pressure, the remaining
foam was used without purification. The procedure generated the
protected L-serine-(.+-.)-Ketoprofen ester as a clear foam (13.72
g, 96% yield). ##STR45##
3-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-2(S)-tert-butoxycarbonylamino-p-
ropionic acid tert-butyl ester
[0415] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.77-7.38 (m,
9H), 5.29 (d, 1/2H, J=6.9 Hz), 5.13 (d, 1/2H, J=6.9 Hz), 4.44-4.30
(m, 3H), 3.78 (q, 1H, J=7 Hz), 1.50 (d, 3H, J=7 Hz), 1.39 (m,
18H).
[0416] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=. 196.13, 173.37,
168.37, 154.99, 140.40, 137.89, 132.50, 131.44, 130.01, 129.25,
129.14, 128.53, 128.29, 82.72, 80.03, 65.22, 64.91, 53.62, 53.40,
45.29, 28.41, 28.02, 27.91, 18.59, 18.50.
[0417] The protected (R,S)-Ketoprofen-L-serine ester (13.6 g,
127.31 mmol) was dissolved in anhydrous hydrochloric acid in
diethyl ether (2M, 100 mL) under an argon atmosphere, at room
temperature. The mixture was allowed to stir for 23 hours at room
temperature when dichloromethane was added (100 mL). After 48
hours, the mixture was concentrated under reduced pressure. The
remaining light yellow foam (9.0 g) was dissolved in a mixture of
dichloromethane (200 mL) and DIUF water (50 mL). After mixing at
room temperature, the layers were separated. The dichloromethane
layer was acidified with 2N hydrochloric acid in ether (5 mL),
dried over sodium sulfate (10 g) filtered and concentrated under
reduced pressure. The remaining foam (6.4 g) was stirred with
dichloromethane (40 mL) for 30 minutes at room temperature under an
argon atmosphere. Diethyl ether was added (20 mL) and the mixture
was allowed to stir for 2 hours at room temperature. After 2 hours,
the solids were filtered and dried under high vacuum at room
temperature until a constant weight was obtained. The experiment
produced L-serine-R,S(.+-.)-Ketoprofen ester, hydrochloride (2.5 g,
22% yield). ##STR46##
2(S)-Amino-3-[2(R,S)-(3-benzoyl-phenyl)-propionyloxy]-propionic
acid, hydrochloride
[0418] H NMR (300 MHz, DMSO): .delta.=8.79 (br s, 3H), 8.72 (br s,
3H), 7.76-7.54 (m, 9H), 4.57 (m, 1H), 4.42-4.28 (m, 2H), 4.01 (m,
1H), 1.46 (d, 3H, J=6 Hz).
[0419] .sup.13C NMR (75 MHz, DMSO): .delta.=195.33, 172.92, 168.01,
167.96, 140.50, 140.39, 136.97 (d), 136.75, 132.66, 131.93 (d),
129.55, 128.65 (d), 128.49 (d), 62.18, 51.35 (d), 44.07, 18.62,
18.41.
[0420] HPLC analysis:
[0421] 98.99% purity; r.t.=9.205 min. (broad peak); 55% DIUF water
(0.1% TFA)/45% methanol; 1 mL/min; 36.4 C; Luna C18, 5 u column
(serial # 211739-42), 4.6.times.250 mm; 20 ul injection.
[0422] CHN analysis:
[0423] calc.: C, 60.40; H, 5.34; N, 3.71. found: C, 60.15; H, 5.32;
N, 3.72.
[0424] Melting Point: 116-120.degree. C. (uncorrected)
Preparation of the (.+-.)Ketoprofen-L-hydroxyproline Ester,
Hydrochloride
[0425] (.+-.)-Ketoprofen (6.70 g, 26.3 mmol),
N-t-butylcarbonyl-trans-L-hydroxyproline-t-butyl ester
(Boc-Hyp-OtBu, 7.40 g, 25.7 mmol, prepared by the literature
method), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,
hydrochloride (EDC, 5.25 g, 27.3 mmol), and
4-(N,N-dimethylamino)-pyridine (DMAP, 0.10 g) were dissolved in
dichloromethane (50 mL) at room temperature, under an argon
atmosphere. After stirring for 3.5 hours, the dichloromethane layer
was washed first with water (50 mL), then, 5% hydrochloric acid
(2.times.25 mL), then water (25 mL), then saturated sodium
bicarbonate (2.times.25 mL), and finally water (50 mL). After
drying for one hour over sodium sulfate (5 g), filtration, and
concentration under reduced pressure, the remaining light green oil
(13.30 g) was purified by column chromatography on silica gel (120
g), eluting with heptane/ethyl acetate (2:1). After combining the
fractions containing product, concentration under reduced pressure
and drying under high vacuum, the procedure generated the protected
L-hydroxyproline-(.+-.)-Ketoprofen ester as a clear oil (5.50 g,
41% yield). ##STR47##
4(R)-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-pyrrolidine-1,2(S)-dicarboxy-
lic acid di-tert-butyl ester
[0426] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.77-7.38 (m,
9H), 5.29 (d, 1/2H, J=6.9 Hz), 5.13 (d, 1/2H, J=6.9 Hz), 4.44-4.30
(m, 3H), 3.78 (q, 1H, J=7 Hz), 1.50 (d, 3H, J=7 Hz), 1.39 (m,
18H).
[0427] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=196.25, 173.43
(d), 171.46 (d), 153.66 (d), 140.35, 138.00, 137.47, 132.55,
131.38, 130.05, 129.13, 128.67, 128.30, 81.55, 80.37 (d), 73.31,
72.48, 58.56, 51.86 (d), 45.43, 36.68 (d), 28.49, 28.18, 18.60.
[0428] The protected (R,S)-Ketoprofen-L-hydroxyproline ester (3.30
g, 6.31 mmol) was dissolved in anhydrous hydrochloric acid in
diethyl ether (2M, 20 mL) under an argon atmosphere at room
temperature. After 72 hours, the mixture was concentrated under
reduced pressure. The remaining light yellow foam (2.6 g) was
dissolved in a mixture of dichloromethane (50 mL) and DIUF water
(10 mL). After mixing at room temperature, the layers were
separated. The dichloromethane layer was acidified with 2N
hydrochloric acid in ether (5 mL) dried over sodium sulfate (5 g)
filtered and concentrated under reduced pressure. The remaining
foam (2 g) was stirred with diethyl ether (20 mL) for 30 minutes at
room temperature under an argon atmosphere. The solids were
filtered and dried under high vacuum at room temperature until a
constant weight was obtained. The experiment produced
L-hydroxyproline-R,S(.+-.)-Ketoprofen ester, hydrochloride (1.2 g,
48% yield). ##STR48##
4(R)-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylic
acid, hydrochloride
[0429] H NMR (300 MHz, DMSO): .delta.=10.25 (br s, 2H), 7.73-7.53
(m, 9H), 5.29 (br m, 1H), 4.38 (t, 1/2H, J=8.1 Hx), 4.26 (t, 1/2H,
J=9 Hz), 3.95 (m, 1H), 3.60 (m, 1H), 3.28 (d, 1/2H, J=13 Hz), 3.16
(d, 1/2H, J=12 Hz), 2.37-2.20 (m, 2H), 1.45 (m, 3H).
[0430] .sup.13C NMR (75 MHz, DMSO): .delta.=195.38, 172.78, 172.73,
169.16, 140.50, 140.41, 137.08, 136.77, 132.67, 132.01, 131.89,
129.52, 128.78, 128.50, 128.50, 72.87 (d), 57.60, 57.52, 50.16 (d),
44.30, 44.20, 34.26, 34.15, 18.43, 18.25.
[0431] HPLC analysis:
[0432] 99.99% purity; r.t.=7.842 and 7.689 min. (broad double
peak); 55% DIUF water (0.1% TFA)/45% methanol; 1 mL/min; 36.4 C;
Luna C18, 5 u column (serial # 211739-42), 4.6.times.250 mm; 20 ul
injection.
[0433] CHN analysis:
[0434] calc.: C, 62.45; H, 5.49; N, 3.47. found: C, 61.78; H, 5.56;
N, 3.62.
[0435] Melting Point: 170-173.degree. C. (uncorrected).
Synthesis of Keotorlac-L-threonine Ester and Human Trials:
Overview:
[0436] The procedure for the synthesis of the L-threonine ester of
Ketorolac is outlined in Synthetic Sequence section. The complete
procedure and analytical data is given in the Experimental Section.
In general, (.+-.)-Ketorolac was extracted from the tromethamine
salt (10 g) and coupled with N-boc-L-threonine t-butyl ester (1
equivalent) with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,
hydrochloride (EDCI) in the presence of a catalytic amount of
4-(N,N-dimethylamino)-pyridine (DMAP). The crude protected
L-threonine-(.+-.)-Ketorolac ester was purified by flash
chromatography. The protecting groups were removed by treatment
with trifluoroacetic acid. The mixture of
L-threonine-R,S(.+-.)-Ketorolac ester salts was separated by
crystallization from acetonitrile/acetone. A sample of
S(-)-Ketorolac L-threonine ester, hydrochloride (2.1 g) separated
from the mixture was shipped to Signature for testing. Synthetic
Sequence: ##STR49## Synthesis of the L-threonine Esters of
(.+-.)-Ketorolac: a) AcOH/H.sub.2O, CH.sub.2Cl.sub.2; b) EDC, DMAP,
CH.sub.2Cl.sub.2; c) TFA; d) HCl, Ethanol; e) ACN-acetone
(Crystallization). Experimental Section:
[0437] The synthesis of SPI0031A was conducted in a single batch.
The procedure was later repeated to ensure reproducibility.
Reagents mentioned in the experimental section were purchased at
the highest obtainable purity from Cayman Chemical, Sigma-Aldrich,
Acros, or Bachem, except for solvents, which were purchased from
either Fisher Scientific or Mallinkrodt.
Preparation and Separation of S(-)-Ketorolac L-threonine Ester,
Hydrochloride (SPI0031A).
[0438] (.+-.)-Ketrolac tromethamine salt (10 g, Cayman Chemical)
was dissolved in water (100 mL), acetic acid (20 mL), and
dichloromethane (50 mL). After mixing for ten minutes, the layers
were separated and the water fraction was extracted two additional
times with dichloromethane (50 mL). The dichloromethane fractions
were combined, dried over sodium sulfate, filtered, concentrated,
and dried under high vacuum at room temperature until a constant
weight was obtained. The procedure generated (.+-.)-Ketrolac (6.78
g, 100% yield) as an off-white solid.
[0439] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=9.62 (1H, br s),
7.80 (2H, d, J=6.9 Hz), 7.55-7.42 (3H, m), 6.84 (1H, d, J=4.0 Hz),
6.15 (1H, d, J=4.0 Hz), 4.62-4.41 (2H, m), 4.10 (1H, dd, J=8.4, 5.7
Hz), 2.97-2.75 (2H, m).
[0440] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=185.25, 176.69,
142.04, 139.05, 131.61, 129.02, 128.26, 127.31, 125.43, 103.63,
47.77, 42.64, 31.20.
[0441] (.+-.)-Ketrolac (6.80 g, 26.6 mmol),
N-tert-butylcarbonyl-L-threonine tert-butyl ester (Boc-Thr-OtBu,
7.33 g, 26.6 mmol, prepared by the literature method),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDCI,
5.50 g, 28.6 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.10
g) were dissolved in dichloromethane (75 mL) at room temperature,
under an argon atmosphere. After stirring for 6 hours, the
dichloromethane solution was washed first with water (50 mL), then
with saturated sodium bicarbonate 50 mL), and finally with water
(50 mL) again. After drying the dichloromethane solution for one
hour over sodium sulfate (10 g), filtration, and concentration
under reduced pressure, the remaining brown oil (14.55 g) was
purified by column chromatography on silica gel (250 g), eluting
with heptane/ethyl acetate (1:1). After combining the fractions
containing product, concentration and drying under high vacuum, the
procedure generated the protected L-threonine-(.+-.)-Ketorolac
ester (SPI003101) as light brown solid foam (13.53 g, 99.2% yield).
##STR50##
[0442] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.80 (2H, m),
7.55-7.42 (3H, m), 6.81 (1H, m), 6.08 (1H,m), 5.47 (1H, m), 5.17
(1H, m), 4.60-4.34 (3H, m), 4.01 (1H, m), 2.90-2.70 (2H, m),
1.48-1.32 (21H, m).
[0443] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=184.85, 169.91,
168.91, 155.84, 141.91, 139.12, 131.41, 128.86, 128.13, 127.18,
124.99, 103.56, 103.13, 82.76, 80.23, 72.15, 72.00, 57.64, 47.62,
42.71, 42.53, 32.02, 31.11, 28.44, 28.02, 17.03, 14.33.
[0444] The protected (.+-.)-Ketorolac L-threonine ester SPI003101
(13.50 g, 26.33 mmol) was dissolved in trifluoroacetic acid (50 mL)
under an argon atmosphere, at room temperature. The mixture was
allowed to stir for 7 hours at room temperature under an argon
atmosphere. The brown solution was concentrated under reduced
pressure and dried under high vacuum at room temperature until a
constant weight was achieved. The remaining brown solid (10.2 g)
was stirred in acetone (250 mL) at room temperature for 3 hours.
The white precipitate that formed was filtered and dried under high
vacuum. The remaining solid (5.70 g) was dissolved in a minimal
amount of DIUF water (5-10 mL) and a 1:1 mixture of
acetonitrile-acetone (100 mL) was added drop-wise over 1 hour while
stirring at room temperature. After the addition was complete, the
mixture was stored for 2 hours at room temperature. The white
precipitate that formed was filtered and dried under high vacuum at
room temperature to a constant weight. The white solid (3.0 g) was
purified a final time by dissolving in DIUF water (5 mL). Most of
the water was removed under reduced pressure to generate a thin,
clear oil. Acetone (100 mL) was added to the oil in a drop-wise
fashion over 30 minutes while stirring under an argon atmosphere.
The mixture was stored for 3 hours at -10.degree. C. The
precipitate was filtered and dried under high vacuum at room
temperature until the weight was constant. The experiment produced
S(-)-Ketorolac-L-threonine ester, hydrochloride SPI0031A (2.32 g,
22.4% yield based on SPI003101, 98.12% purity by HPLC) as a white
solid.
[0445] .sup.1H NMR (300 MHz, DMSO): .delta.=8.80 (3H, br s), 7.73
(2H, d, J=7.5 Hz), 7.61-7.46 (3H, m), 6.77 (1H, d, J=3.9 Hz), 6.16
(1H, d, J=3.9 Hz), 5.33 (1H, m), 4.42-4.22 (4H, m), 2.76 (2H, m),
1.35 (3H, d, J=6.6 Hz).
[0446] .sup.13C NMR (75 MHz, DMSO): .delta.=183.41, 169.64, 168.19,
142.32, 138.59, 131.44, 128.35, 128.26, 126.22, 124.27, 103.18,
68.84, 55.31, 47.34, 41.83, 30.18, 16.59.
[0447] CHN analysis:
[0448] calc.: C, 58.09; H, 5.39; N, 7.13; Cl, 9.02.
(C.sub.19H.sub.21ClN.sub.2O.sub.5); found: C, 58.61; H, 5.26; N,
7.10; Cl, 8.16.
[0449] HPLC analysis:
[0450] 98.12% purity, r.t.=19.617 min, sample dissolved in DIUF
water/ACN, 50% DIUF water (0.1% TFA)/50% ACN, Gemini C18
(#262049-2), 5 u, 250.times.4.6 mm, 1 mL/min., 37.degree. C., 20 uL
inj. vol., SPD-10 Avp, chl-210 nm.
[0451] Specific rotation: -108 deg (25.degree. C., 52.5 mg/5 mL
water, 589 nm)
[0452] Melting Point: 155-157.degree. C. (decomposed)
Large negative specific rotation is consistent with the
S(-)Ketorolac moiety.
Human Clinical Trial:
[0453] S(-)Ketorolac-L-Threonine ester capsules were filled using
dextrose as the filler. The Ketorolac-L-Threonine ester dose was
comparable to racemic Ketorolac Tromethamine tablets. For example,
13 mg ot S(-)Ketorolac-L-Threonine ester was roughly equivalent to
13 mg of racemic Ketorolac tromethamine in tablet and/or capsule
form.
[0454] A Female patient (age 72) suffereing from severe ankolysing
spodolytis, arthritis and other inflammatory joint probems was
under treatment with Indomethacin, 25 mg twice daily dose. In order
the evaluate the analgesic activity of S(-) Ketorolac-L-Threonine
ester, the patient was withdrawn from Indomethacin. After 24 hours,
the pain returned and she was administered with 13 mg of S(-)
Ketorolac L-Threonine ester, once in the morning and once in the
evening. This was repeated for 5 days. During the entire period,
the patient demonstrated lack of pain, no gastric irritation
symptoms, or other side effects.
[0455] About 3 months later, the same above female volunteer
repeated the experiment. This time, after she went off the
indomethacin, only one dose of 13 mg of S(-) Ketorolac-L-Threonine
ester was administered. After the 2.sup.nd day, she complained of
the pain resurfacing, and the dose was then increased on the
morning of the 3.sup.rd day to twice daily of 13 mg each. Beginning
the 3.sup.rd day she indicated of lack of any pain. This treatment
was continued for another 3 days of two capsules of 13 mg each. On
the morning of the 6.sup.th day, she was switched back to
Indomethacin. This study showed that in this particular volunteer,
13 mg twice daily was the appropriate dose to alleviate her severe
pain.
C. Amino Acid Derivaties of Aspirin
Overview:
[0456] The procedure for the synthesis of the L-serine,
L-threonine, and L-hydroxyproline esters of acetylsalicylic acid is
outlined in Synthetic Sequence section and is exemplary for other
amino acids. The complete procedure and analytical data is given in
the Experimental Section. In general, acetylsalicyloyl chloride (10
g-25 g, in batches) was coupled with the N-benzyloxy/benzyl ester
protected amino acids in the presence of pyridine. Once the
reactions were completed (24 to 48 hours at room temperature), the
mixture was poured into ice-cold 2N hydrochloric acid. The
dichloromethane fraction was then washed with sodium bicarbonate,
water and brine. After drying over sodium sulfate, filtration, and
concentration, the crude protected amino acid esters of
acetylsalicylic acid were purified by flash chromatography on
silica gel. The procedure generated the protected amino acid esters
of acetylsalicylic acid in yields ranging from 68% to 95%. The
protecting groups were removed by hydrogenation (20 psi H.sub.2) in
the presence of 10% palladium on carbon. The amino acid esters of
acetylsalicylic acid were extracted from the palladium catalyst
using water. The solution containing the product uses concentrated,
and dried. The final compounds was washed with solvent (water,
dioxane, acetonitrile, and/or dichloromethane) until pure and dried
under high vacuum until a constant weight was achieved. Synthetic
Sequence ##STR51## ##STR52## Synthesis of the L-serine,
L-threonine, and L-hydroxyproline Esters of Acetylsalicylic Acid:
a) Pyridine, CH.sub.2Cl.sub.2; b) 10% Pd/C, EtOH, EtOAc.
Experimental Section:
[0457] The synthesis of SPIB00101, SPIB00102 and SPIB00103 was
conducted in one or two batches. Reagents mentioned in the
experimental section were purchased at the highest obtainable
purity from Lancaster, Sigma-Aldrich, Acros, or Bachem, except for
solvents, which were purchased from either Fisher Scientific or
Mallinkrodt.
1) SPIB00102: 2-O-Acetylsalicylic Acid (2S,3R)-(-)-threonine
Ester
[0458] A mixture of N-carbobenzyloxy-L-threonine benzyl ester
(Z-Thr-OBzl, 21.77 g, 63.40 mmole) and pyridine (25 mL) in
anhydrous dichloromethane (500 mL) was cooled in an ice bath while
under a nitrogen atmosphere. Acetylsalicyloyl chloride (17.63 g,
88.76 mmole) was added, and the mixture was allowed to warm to room
temperature and stir overnight. After 24 hours, the mixture was
poured into ice-cold 2N hydrochloric acid (400 mL). After mixing,
the layers were separated and the dichloromethane fraction was
washed first with water (500 mL), then saturated sodium bicarbonate
solution (500 mL), then water (500 mL), then brine (500 mL) and
dried over sodium sulfate (25 g). After filtration, concentration
under reduced pressure, and drying under high vacuum, the remaining
yellow oil (35.43 g) was purified by flash chromatography on silica
gel (300 g, 0.035-0.070 mm, 6 nm pore diameter), eluting with
hexanes/ethyl acetate (3:1). After concentration of the product
containing fractions under reduced pressure and drying under high
vacuum until the weight was constant, the experiment produced the
protected acetylsalicylic-L-threonine ester SPIB0010201 (28.1 g,
88% yield) as a colorless oil. ##STR53##
[0459] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.74 (1H, d,
J=7.5 Hz), 7.51 (1H, dt, J=7.5, 1.5 Hz), 7.34-7.17 (11H, m), 7.06
(1H, d, J=7.2 Hz), 5.62 (2H, m), 5.13 (4H, m), 4.65 (1H, dd, J=9.6,
2.4 Hz), 2.29 (3H, s), 1.38 (3H, d, J=6.6 Hz).
[0460] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=169.35, 169.22,
162.73, 156.26, 150.41, 135.79, 134.67, 133.77, 131.24, 128.35,
128.24, 128.08, 127.95, 125.78, 123.51, 122.61, 71.22, 67.72,
67.26, 57.64, 20.98, 16.88.
[0461] The protected acetylsalicylic-L-threonine ester SPIB0010201
(14.50 g, 28.68 mmole) was dissolved in ethanol (100 mL) and ethyl
acetate (100 mL) at room temperature and added to a Parr bottle
that contained 10% palladium on carbon (3.0 g, 50% wet) under a
nitrogen atmosphere. The nitrogen atmosphere was replaced with
hydrogen gas (20 psi). After 20 hours of shaking, the palladium
catalyst was removed by filtration through celite. The remaining
solids (palladium/celite and product) were washed with water
(600.times.4 mL) until the product was removed. The ethanol and
water fractions were concentrated under reduced pressure at room
temperature. The remaining solids were washed with water (20 mL)
and dioxane (20 mL) for 48 hours. After filtration, the remaining
white solid was dried at room temperature under high vacuum until
the product weight was constant (16 hours). The experiment produced
acetylsalicylic-L-threonine ester, SPIB00102 (4.40 g, 55% yield) as
a white solid. ##STR54##
[0462] .sup.1H NMR (300 MHz, D.sub.2O-DCl): .delta.=8.00 (1H, dd,
J=7.8, 1.5 Hz), 7.74 (1H, dt, J=7.8, 1.5 Hz), 7.47 (1H, dt, J=7.8,
1.5 Hz), 7.27 (1H, dd, J=7.8, 1.5 Hz), 5.76 (1H, dq, J=6.9, 3.0
Hz), 4.49 (1H, d, J=3.0 Hz), 2.39 (3H, s), 1.55 (3H, d, J=6.9
Hz).
[0463] .sup.13C NMR (75 MHz, D.sub.2O-DCl): .delta.=173.03, 168.84,
163.97, 149.56, 135.32, 131.26, 126.85, 123.48, 121.49, 69.16,
56.36, 20.45, 15.86.
[0464] HPLC analysis:
[0465] 98.7% purity; rt=6.233 min; Luna C18 5 u column (sn
167917-13); 4.6.times.250 mm; 254 nm; 35% MeOH/65% TFA (0.1%)
pH=1.95; 35 C; 20 ul inj.; 1 ml/min; sample dissolved in mobile
phase with 1 drop phosphoric acid.
[0466] CHN analysis:
[0467] calc.: C, 55.51; H, 5.38; N, 4.98. found: C, 55.37; H, 5.40;
N, 5.03.
[0468] Melting point: 153.5.degree. C. (dec.)
2) SPIB00101: 2-O-Acetylsalicylic Acid (2S)-(+)-serine Ester
[0469] A mixture of N-carbobenzyloxy-L-serine benzyl ester
(Z-Ser-OBzl, 23.17 g, 70.34 mmole) and pyridine (30 mL) in
anhydrous dichloromethane (500 mL) was cooled in an ice bath while
under a nitrogen atmosphere. Acetylsalicyloyl chloride (21.07 g,
106.1 mmole) was added and the mixture was allowed to warm to room
temperature and stir over two days. After 48 hours, the mixture was
poured into ice-cold 2N hydrochloric acid (400 mL). After mixing,
the layers were separated and the dichloromethane fraction was
first washed with water (500 mL), then saturated sodium bicarbonate
solution (500 mL), then water (500 mL), then brine (500 mL) and
dried over sodium sulfate (25 g). After filtration, concentration
under reduced pressure, and drying under high vacuum, the remaining
brown solid (47.19 g) was purified by flash chromatography on
silica gel (200 g, 0.035-0.070 mm, 6 nm pore diameter), eluting
with hexanes/ethyl acetate (3:1). After concentration of the
product containing fractions under reduced pressure and drying
under high vacuum until the weight was constant, the experiment
produced the protected acetylsalicylic-L-serine ester SPIB0010101
(32.97 g, 95% yield) as a white solid. ##STR55##
[0470] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.74 (1H, d,
J=7.8 Hz), 7.55 (1H, dt, J=7.8, 1.5 Hz), 7.33-7.21 (11H, m), 7.08
(1H, d, J=7.5 Hz), 5.68 (1H, d, J=8.4 Hz), 5.20 (2H, s), 5.12 (2H,
s), 4.77 (1H, m), 4.66 (1H, dd, J=11.4, 3.3 Hz), 4.57 (1H, dd,
J=11.4, 3.3 Hz), 2.30 (3H, s).
[0471] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=169.45, 169.09,
163.68, 163.35, 155.57, 150.77, 135.87, 134.75, 134.07, 131.44,
128.50, 128.43, 128.27, 128.14, 128.04, 125.92, 123.71, 122.18,
67.83, 67.27, 64.63, 53.55, 21.03.
[0472] The protected acetylsalicylic-L-serine ester SPIB0010101
(21.0 g, 42.7 mmole) was dissolved in ethanol (100 mL) and ethyl
acetate (100 mL) at room temperature and added to a Parr bottle
that contained 10% palladium on carbon (4.20 g, 50% wet) under a
nitrogen atmosphere. The nitrogen atmosphere was replaced with
hydrogen gas (20 psi). After 5 hours additional 10% palladium
catalyst (4.26 g) was added and the hydrogen atmosphere was
returned (20 psi). After an additional 20 hours of shaking at room
temperature, the palladium catalyst was removed by filtration
through celite. The remaining solids (palladium/celite and product)
were washed with water (1500.times.2 mL) until the product was
removed. The ethanol and water fractions were concentrated under
reduced pressure at room temperature. The remaining solid (7.17 g)
was dissolved in DIUF water (4.3 L), filtered through celite to
remove insoluble material, and concentrated under high vacuum at
room temperature. The white solid was then washed with 1,4-dioxane
(100 mL) and DIUF water (50 mL) overnight. After 24 hours the solid
was filtered and dried under high vacuum until the weight was
constant (24 hours). The experiment produced the
acetylsalicylic-L-serine ester SPIB00101 (6.17 g, 54% yield) as a
white solid. ##STR56##
[0473] .sup.1H NMR (300 MHz, D.sub.2O-DCl): .delta.=8.05 (1H, dd,
J=7.8, 1.5 Hz), 7.75 (1H, dt, J=7.8, 1.5 Hz), 7.47 (1H, dt, J=7.8,
0.9 Hz), 7.27 (1H, dd, J=7.8, 0.9 Hz), 4.87 (1H, dd, J=12.6, 4.2
Hz), 4.79 (1H, dd, J=12.6, 3.0 Hz), 4.62 (1H, dd, J=4.2, 3.0 Hz),
2.39 (3H, s).
[0474] .sup.13C NMR (75 MHz, D.sub.2O-DCl): .delta.=173.01, 168.58,
164.54, 149.72, 135.39, 131.59, 126.87, 123.62, 121.15, 62.38,
52.05, 20.44.
[0475] HPLC analysis:
[0476] 98.1% purity; r.t.=5.839 min.; 65% TFA (0.1%)/35% methanol;
1 mL/min; 35 C; Luna C18, 3 u column (SN 184225-37), 4.6.times.250
mm; 22 ul injection; DAD1B, Sig=240, 4 Ref=550,100.
[0477] CHN analysis:
[0478] calc.: C, 53.93; H, 4.90; N, 5.24. found: C, 54.02; H, 5.00;
N, 5.23.
[0479] Melting point: 147.0.degree. C. (dec.)
3) SPIB00103: 2-O-Acetylsalicylic Acid (2S,4R)-4-hydroxyproline
Ester
[0480] A mixture of N-carbobenzyloxy-L-hydroxyproline benzyl ester
(Z-Ser-OBzl, 21.5 g, 60.5 mmole) and pyridine (25 mL) in anhydrous
dichloromethane (500 mL) was cooled in an ice bath while under a
nitrogen atmosphere. Acetylsalicyloyl chloride (13.2 g, 66.6 mmole)
was added and the mixture was allowed to warm to room temperature
and stir overnight. After 24 hours, additional acetylsalicyloyl
chloride (5.0 g, 25.2 mmole) was added and the mixture was allowed
to stir overnight. After 48 hours, the mixture was poured into
ice-cold 1N hydrochloric acid (500 mL). After mixing, the layers
were separated and the dichloromethane fraction was washed with
water (500 mL), then saturated sodium bicarbonate solution (500
mL), then water (500 mL), then brine (500 mL) and dried over sodium
sulfate (25 g). After filtration, concentration under reduced
pressure, and drying under high vacuum, the remaining yellow oil
(40.7 g) was purified by flash chromatography on silica gel (460 g,
0.035-0.070 mm, 6 nm pore diameter), eluting with heptane/ethyl
acetate (3:1). After concentration of the fractions containing
product under reduced pressure and drying under high vacuum until
the weight was constant, the experiment produced the protected
acetylsalicylic-L-hydroxyproline ester SPIB0010301 (21.31 g, 68%
yield) as a colorless oil. ##STR57##
[0481] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.92 (1H, d,
J=7.8 Hz), 7.56 (1H, t, J=7.8 Hz), 7.34-7.21 (10H, m), 7.09 (1H, d,
J=7.8 Hz), 5.48 (1H, s), 5.21 (2H, m), 5.03 (2H, d, J=15 Hz), 4.57
(1H, m), 3.85 (2H, m), 2.53 (1H, m), 2.28 (4H, m).
[0482] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=171.72, 171.49,
169.25, 163.47, 163.30, 154.52, 153.93, 150.54, 136.05, 135.94,
135.21, 135.00, 134.17, 134.12, 128.43, 128.32, 128.28, 128.20,
128.05, 127.98, 127.94, 127.79, 125.89, 123.70, 122.46, 122.38,
73.24, 72.59, 67.33, 67.11, 66.97, 58.02, 57.69, 52.47, 52.15,
36.74, 35.65, 20.90.
[0483] The protected acetylsalicylic-L-hydroxyproline ester
SPIB0010301 (10.6 g, 20.5 mmole) was dissolved in ethanol (75 mL)
and ethyl acetate (75 mL) at room temperature and added to a Parr
bottle that contained 10% palladium on carbon (3.0 g, 50% wet)
under a nitrogen atmosphere. The nitrogen atmosphere was replaced
with hydrogen gas (20 psi). After 17 hours of shaking at room
temperature, the reaction mixture was washed with water (500 mL)
for two hours. The organic layer (top) was removed via pipette and
the aqueous layer was filtered through celite. The water fraction
was concentrated under reduced pressure at room temperature. The
remaining solid (6.71 g) was then washed with anhydrous
dichloromethane (35 mL) overnight. After 24 hours the solid was
filtered and dried under high vacuum until the weight was constant
(24 hours). The experiment produced
acetylsalicylic-L-hydroxyproline ester, SPIB00301 (2.87 g, 47.7%
yield) as a white solid. ##STR58##
[0484] .sup.1H NMR (300 MHz, D.sub.2O-DCl): .delta.=8.09 (1H, d,
J=7.5 Hz), 7.75 (1H, t, J=7.5 Hz), 7.48 (1H, t, J=7.5 Hz), 7.28
(1H, d, J=7.5 Hz), 5.69 (1H, m), 4.76 (1H, t, J=7.5 Hz), 3.86 (1H,
dd, J=13.5, 3.9 Hz), 3.74 (1H, d, J=13.5 Hz), 2.81 (1H, dd, J=15.0,
7.5 Hz), 2.60 (1H, m), 2.40 (3H, s).
[0485] .sup.13C NMR (75 MHz, D.sub.2O-DCl): .delta.=173.13, 170.25,
164.31, 149.65, 135.36, 131.54, 126.87, 123.54, 121.37, 73.86,
58.34, 50.95, 34.38, 20.48.
[0486] HPLC analysis:
[0487] 98.3% purity; r.t.=7.201 min.; 65% TFA (0.1%)/35% methanol;
1 mL/min; 35 C; Luna C18, 3 u column (SN 184225-37), 4.6.times.250
mm; 22 ul injection; DAD1B, Sig=240, 4 Ref=550,100.
[0488] CHN analysis:
[0489] calc.: C, 57.34; H, 5.16; N, 4.78. found: C, 57.09; H, 5.23;
N, 4.91.
[0490] Melting point: 162.degree. C. (dec.)
Comparsion of the L-serine, L-threonine, and L-Hydroxyproline
Esters of Acetylsalicylic Acid to Acetylsalicylic Acid with Respect
to Gastric Mucosa Irritation
[0491] The present study was conducted to determine the relative
potential of the new formulations of aspirin (L-serine,
L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid)
to cause gastric mucosal irritation/lesions in fasted male albino
rats. Aspirin served as a reference control.
[0492] Different new formulations of aspirin and aspirin were
administered by gavage to fasted male albino rats (Wistar strain),
using 0.5% (w/v) Carboxymethylcellulose (CMC) in Phosphate Buffer
(pH 2.6) solution as the vehicle. The study was conducted at two
dose levels viz. 100 mg and 200 mg/kg body weight along with a
vehicle control group. At each dose level 5 animals were used. All
the doses were expressed as aspirin molar equivalents. The doses
used as well as the molar equivalents are presented below.
TABLE-US-00020 TABLE 16 Formulation: Molar equivalent Formulation
Molar equivalent L-serine ester of 1.483 units are equivalent to 1
unit of aspirin acetylsalicylic acid L-Hydroxyproline ester 1.628
units are equivalent to 1 unit of aspirin of acetylsalicylic acid
L-threonine ester of 1.561 units are equivalent to 1 unit of
aspirin. acetylsalicylic acid
[0493] TABLE-US-00021 TABLE 17 Test Item: Group: Dose (mg per kg)
[in terms of acetylsalicylic acid]: Equivalent weight of the Test
item [mg] Dose (mg per kg) [in terms of Equivalent acetylsalicylic
weight of the Test Item Group acid] Test item [mg] Vehicle control
Vehicle control 0.0 -- Group L-serine ester of Test Group 1 100.0
148.3 acetylsalicylic acid L-serine ester of Test Group 1 100.0
148.3 acetylsalicylic Test Group 2 200.0 296.6 acid
L-Hydroxyproline Test Group 1 100.0 162.8 ester of Test Group 2
200.0 325.6 acetylsalicylic acid L-threonine, ester Test Group 1
100.0 156.1 of acetylsalicylic Test Group 2 200.0 312.2 acid
Reference control Test Group 1 100.0 100.0 acetylsalicylic Test
Group 2 200.0 200.0 acid
[0494] The rats were fasted for a period of 18 to 22 hours before
dosing. The test item was administered as a single dose by gavage.
Three hours after drug administration, the animals were killed
humanely by CO.sub.2 gas inhalation. The stomach was dissected out
and observed for [0495] the quantity of mucous exudate, [0496]
degree of hyperemia and thickening of stomach wall, [0497]
hemorrhagic spots (focal or diffuse), nature of hemorrhages
(petechial or ecchymotic) along with the size and [0498]
perforations
[0499] The observations on gastric mucosal irritation of animals of
various groups are summarized below: TABLE-US-00022 TABLE 18 Test
Item: Group: Dose mg/kg (as acetylsalicylic acid): Observation Dose
mg/kg (as acetylsalicylic Test Item Group acid Observation Vehicle
control Vehicle 0.0 None of the animals showed control Group any
evidence of gastric mucosal irritation L-serine ester of Test Group
1 100.0 None of the dosed animals acetylsalicylic acid showed any
evidence of gastric mucosal irritation Test Group 2 200.0 None of
the dosed animals showed any evidence of gastric mucosal
irritation. L-Hydroxyprolin ester Test Group 1 100.0 None of the
dosed animals of acetylsalicylic acid showed any evidence of
gastric mucosal irritation. Test Group 2 200.0 None of the dosed
animals showed any evidence of gastric mucosal irritation
L-threonine, ester of Test Group 1 100.0 None of the dosed animals
acetylsalicylic acid showed any evidence of gastric mucosal
irritation Test Group 2 200.0 None of the dosed animals showed any
evidence of gastric mucosal irritation Reference control Test Group
1 100.0 None of the dosed animals (acetylsalicylic acid) showed any
evidence of gastric mucosal irritation Test Group 2 200.0 All the 5
animals dosed, showed evidence of gastric mucosal irritation.
[0500] In conclusion it was observed that none of the L-serine,
L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid
induced any evidence of irritation of gastric mucosa at the two
doses tested viz., 100 and 200 mg/kg body weight. In contrast,
aspirin (acetylsalicylic acid) caused irritation of the gastric
mucosal in all the fasted male albino rats at the dose level of 200
mg/kg. However at the dose level of 100 mg/kg aspirin failed to
cause any evidence of gastric mucosal irritation in the male
rats.
Further none of the animals of different test groups showed any
clinical symptoms of toxicity during the observation period of
three hours.
Efficacy of L-serine, L-threonine, and L-Hydroxyproline Esters of
Acetylsalicylic Acid Compared to Acetylsalicylic Acid on Clotting
Time in Rats
Observations of Blood Clotting Time
[0501] The data on the mean clotting time (MCT) of the animals of
low, intermediate and high dose groups of different formulations,
vehicle control and positive control groups estimated one hour
after dosing were presented below (Table 19): TABLE-US-00023 TABLE
19 Summary of Mean Clotting Time (.+-.S.D.) in Minutes - L-serine,
L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid
and Aspirin (Positive control): Low dose: Intermediate dose: High
dose Low Dose Intermediate Dose High Dose Vehicle control 4.9 .+-.
1.10 L-serine ester of 5.7 .+-. 1.34 6.8 .+-. 1.48 6.9 .+-. 1.37
acetylsalicylic acid L-Hydroxyprolin ester 6.1 .+-. 1.10 5.7 .+-.
0.82 7.5 .+-. 1.18 of acetylsalicylic acid L-threonine, ester of
5.2 .+-. 1.14 5.6 .+-. 0.84 7.4 .+-. 0.97 acetylsalicylic acid
Positive control 6.2 .+-. 1.40 8.1 .+-. 1.97 9.8 .+-. 1.32
(acetylsalicylic acid)
FIG. 3-6 depict the group mean data of animals regarding the dose
relationship+mean clotting time in minutes for the L-series ester
of aspirin and for the control.
[0502] The statistical analysis showed a significant improvement at
5% significance level in the efficacy for the high dose and mid
dose when compared to the vehicle control group (FIG. 7).
[0503] FIG. 4 shows the group mean data of animals. It provides the
dose response relationship to mean clotting time (MCT) in minutes
with respect to L-hydroxyproline ester of aspirin. The statistical
analysis of FIG. 4 showed a significant improvement at 5%
significance level in the efficacy for the high dose and low dose
when compared to the vehicle control group (FIG. 6)
[0504] FIG. 5 depicts the dose response relationship to mean
clotting time (MCT) in minutes of L-threonine ester of
acetylsalicylic acid. The statistical analysis showed a significant
improvement at 5% significance level in the efficacy for the high
dose when compared to the vehicle control.
[0505] FIG. 6 depicts the dose response relationship to mean
clotting time for acetylsalicylic acid. The statistical analysis
showed a significant improvement at 5% significance level in the
efficacy for the intermediate and high dose when compared to the
vehicle control. The dose response effect were statistically
significant and clearly evident (FIG. 7).
Conclusion
[0506] The present study was conducted to evaluate the efficacy of
new formulations of aspirin using blood clotting time as an index
in albino rats. Aspirin served as positive control. The study was
conducted at three dose levels with the new formulations and
positive control along with a vehicle control group.
Doses
[0507] The doses for the main study were selected based on the dose
range finding experiments with acetylsalicylic acid. All the doses
were expressed as aspirin molar equivalents. The doses used are
presented below. TABLE-US-00024 TABLE 20 Test Item: Low Dose
(mg/kg): Intermediate dose 9 mg/kg): High dose (mg/kg) Low Dose
Intermediate High Dose Test Item (mg/kg) Dose (mg/kg) (mg/kg)
L-serine ester of 1.0 4.0 10.0 acetylsalicylic acid L-Hydroxyprolin
ester of 1.0 4.0 10.0 acetylsalicylic acid L-threonine, ester of
1.0 4.0 10.0 acetylsalicylic acid Aspirin (Positive control) 1.0
4.0 10.0
Efficacy (Blood Clotting Time)
[0508] The efficacy in terms of time required for the blood
clotting time at different dose levels--low, intermediate and high
dose for different formulations of various amino acid derivatives
of aspirin and acetylsalicylic acid are presented below.
TABLE-US-00025 TABLE 21 Low dose: Intermediate dose: High dose Low
Dose Intermediate Dose High Dose Vehicle control 4.9 .+-. 1.10
L-serine ester of 5.7 .+-. 1.34 6.8 .+-. 1.48 6.9 .+-. 1.37
acetylsalicylic acid L-Hydroxyprolin ester 6.1 .+-. 1.10 5.7 .+-.
0.82 7.5 .+-. 1.18 of acetylsalicylic acid L-threonine, ester of
5.2 .+-. 1.14 5.6 .+-. 0.84 7.4 .+-. 0.97 acetylsalicylic acid
Positive control 6.2 .+-. 1.40 8.1 .+-. 1.97 9.8 .+-. 1.32
[0509] L-serine, L-threonine, and L-Hydroxyproline esters of
acetylsalicylic acid are as effective as acetylsalicylic acid with
respect to clotting time observed after one hour after
administration but are far superior in terms of the absence of
gastric irritation at all levels compared to acetylsalicylic
acid.
Efficacy of L-serine, L-threonine, and L-Hydroxyproline Esters of
Acetylsalicylic Acid Compared to Acetylsalicylic Acid on Clotting
Time in Rats Estimated Two Hours after Dosing
[0510] The present study was conducted to evaluate the efficacy of
L-serine, L-threonine, and L-Hydroxyproline esters of
acetylsalicylic acid compared to acetylsalicylic acid using blood
clotting time, estimated 2 hours (.+-.10 minutes) after dosing, as
an index in albino rats. Aspirin served as positive control. Male
albino rats were exposed to aspirin and to 3 new formulations of
amino acid derivatives of aspirin derivatives at one dose level of
20 mg/kg body weight. No vehicle control group was used. The doses
were expressed as aspirin molar equivalents. The doses used for the
main experiment for different formulations and positive control was
presented below. TABLE-US-00026 TABLE 22 Test Item: Dose in terms
of Acetylsalicylic acid 9 mg/kg) Dose in terms of Acetylsalicylic
Test Item acid (mg/kg) L-serine ester of acetylsalicylic acid 20.0
L-Hydroxyproline ester of 20.0 acetylsalicylic acid L-threonine
ester of acetylsalicylic acid 20.0 Aspirin (Positive control)
20.0
Efficacy (Blood Clotting Time)
[0511] The efficacy in terms of time required for the blood
clotting time at the dose level of 20 mg/kg body weight for
different formulations of amino acid derivatives of aspirin and
aspirin (positive control) are presented below:
Observations of Blood Clotting Time
[0512] The data on the mean clotting time (MCT) of the animals,
estimated 2 hours (.+-.10 minutes) after dosing, at the dose level
of 20 mg/kg body weight for the formulations of aspirin
derivatives, vehicle control and positive control are presented
below TABLE-US-00027 TABLE 23 Summary of Mean Clotting Time (.+-.
S.D.) in Minutes of L-serine, L-threonine, and L-Hydroxyproline
esters of acetylsalicylic acid compared to acetylsalicylic acid
(Positive control) Dose (20 mg/kg) L-serine ester of 3.8 .+-. 0.92
acetylsalicylic acid L-Hydroxyproline 4.2 .+-. 1.32 ester of
acetylsalicylic acid L-threonine ester of 5.3 .+-. 1.06
acetylsalicylic acid Positive control 5.4 .+-. 1.17
(acetylsalicylic acid)
L-serine, L-threonine, and L-Hydroxyproline esters of
acetylsalicylic acid were found to be effective on clotting
time.
[0513] In conclusion, it was observed that based on the time
required for the blood to clot (clotting time), when estimated 2
hours after dosing, the amino acid derivatives were efficacous.
However, the L-threonine ester of acetylsalicyclic acid was found
to have relatively better efficacy than the other two
formulations.
[0514] As shown by FIG. 7 the statistical analysis showed that
L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid
are at least as effective as acetylsalicylic acid. There is no
significant difference at 5% significance level for
L-Hydroxyproline ester of acetylsalicylic acid and L-threonine
ester of acetylsalicylic with respect to positive control for the
mean blood clotting time observed after two hours. However,
combined with the gastric irritation potential, the L-serine,
L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid
are far superior.
[0515] The following additional data were obtained, when the same
drugs were compared in rats on a different date with different time
intervals and doses: TABLE-US-00028 TABLE 24 Clotting Time (min)
Dose ASA-Ser ASA-Hyp ASA-Thr ASA Vehicle 10 mg/kg 1 hr 6.9 7.5 7.4
9.8 4.9 10 mg/kg 24 hr4.4 3.3 3.6 4.4 2.7 20 mg/kg 2 hr 3.8 4.2 5.3
5.4 2.7 *ASA is acetylsalicyclic acid
Two of the better esters of ASA at various conditions are as
follows: 10 mg/kg 1 hr ASA-Hyp and ASA-Thr 10 mg/kg 24 hr
ASA-Serine and ASA-Thr 20 mg/kg 2 hr ASA-Hyp and ASA-Thr
[0516] When compared against the various amino acid esters,
L-Threonine Ester of Aspirin seems to be the most preferred as it
showed consistently a better response in the rat blood clotting
time.
[0517] Based upon consistently improved toxicity profile, efficacy
and better therapeutic index, Acetylsalicylic Acid-L-Threonine
Ester was advanced to GMP synthesis.
Results of the 28-Day Chronic Dosing in Rodents, Comparative
Toxicology:
[0518] The purpose of this study is to establish the toxicity of
Acetylsalicylic Acid-L-Threonine Ester in relation to Aspirin
(Make: Sigma, Batch number 090K0884) which served as a reference
drug by conducting a 28-day repeated dose oral toxicity test in
male and female albino rats.
[0519] Aspirin and Acetylsalicylic Acid-L-Threonine Ester were
administered to albino rats (Wistar strain), by oral gavage daily
for a period of 28 days, using 0.5% Carboxymethylcellulose (CMC) in
phosphate buffer solution (pH 2.6) as vehicle. The study was
conducted at one dose level only along with a vehicle control group
as per the recommendation of the Sponsor. The test doses are
expressed as Aspirin molar equivalents. Acetylsalicylic
Acid-L-Threonine Ester was compared against Aspirin and Vehicle at
100 mg/kg dose administered to rats for 28 days.
[0520] The salient features of the study are as follows, where
ASA-T represents Acetylsalicylic Acid-L-Threonine Ester: [0521] 1.
All the animals of vehicle control group and the test group
(Acetylsalicylic Acid-L-Threonine Ester) and reference control
group (Aspirin) survived through the dosing period of 28 days.
[0522] 2. None of the animals of the vehicle control group, test
group (Acetylsalicylic Acid-L-Threonine Ester), and reference
control group (Aspirin) exhibited any clinical symptoms of toxicity
through out the dosing period.
[0523] 3. The Changes in the Body weight is tabulated hereinbelow.
TABLE-US-00029 TABLE 25 Body Weight Comparison Significance[P <
0.05] Gain ASA-T vs Aspirin vs Vehicle Normal (Male) Gain ASA-T and
Aspirin vs Vehicle Decrease (Female)
[0524] The percentage decrease were 29% and 21% for Acetylsalicylic
Acid-L-Threonine Ester and Aspirin [0525] 4. Food intake of the
animals of both the sexes of test group (Acetylsalicylic
Acid-L-Threonine Ester) and reference control group (Aspirin) was
found to be normal and comparable to the animals of vehicle control
group.
[0526] 5. Results of hematological analysis of the animals of
different groups are shown in Table 26 below: TABLE-US-00030 TABLE
26 Significance Hematological Comparison [P < 0.05] All Blood
Parameters ASA-T vs Aspirin vs Vehicle None Platelet Count ASA-T vs
Aspirin Increase (Male)
[0527] 6. Results of clinical chemistry analysis of the animals of
different groups are summarized below in Table 27: TABLE-US-00031
TABLE 27 Significance Clinical Chemistry Comparison [P < 0.05]
Alkaline Phosphatase AST-T Ester vs Vehicle Increase (Female) Total
Protein ASA-T Ester .RTM. vs Vehicle Increase (Female) Creatinine
ASA-T Ester .RTM. vs Vehicle Decrease (Male) Cholesterol ASA-T
Ester .RTM. vs Vehicle Increase (Male) Alkaline Phosphatase Aspirin
vs Vehicle Increase (Female) Sodium Aspirin vs Vehicle Increase
(Female) Blood Urea Aspirin vs Vehicle Decrease (Female) SGPT and
Cholesterol Aspirin vs Vehicle Increase (Male) All Clinical
Chemistry ASA-T Ester .RTM. vs Aspirin None(but two below) Blood
Glucose ASA-T Ester .RTM. vs Aspirin Decrease (Female) Creatinine
ASA-T Ester .RTM. vs Aspirin Decrease (Male)
[0528] 7. Necropsy of the surviving animals at the end of the study
(terminal necropsy) of vehicle control and different treatment
groups did not reveal any gross pathological changes in any of the
vital organs. Further there is no evidence of gastric mucosal
irritation in the animals of vehicle control, test group (ASA-T
Ester) and reference control group (Aspirin).
[0529] 8. The data on absolute (Abs) and relative (Rel) organ
weights of liver, kidney, adrenals, heart, spleen and testes showed
the following changes in the organ weights are depicted in Table
28: TABLE-US-00032 TABLE 28 Rel/Abs Organ Wts Comparison
Significance[P < 0.05] Adrenals (Abs) ASA-T Ester .RTM. vs
Vehicle Decrease (Male) Kidney (Abs) Aspirin vs Vehicle Decrease
(Male) Spleen (Abs) Aspirin vs Vehicle Decrease (Female) Kidney
(Rel) Aspirin vs Vehicle Increase (Male) Spleen (Rel) Aspirin vs
Vehicle Increase (Female) Kidney (Abs) ASA-T Ester vs Aspirin
Decrease (Male) Spleen (Abs) ASA-T Ester vs Aspirin Decrease (Male)
Kidney (Rel) ASA-T Ester vs Aspirin Increase (Male)
[0530] 9. Histological sections of the following organs viz. brain,
stomach, small intestines, large intestine, liver, kidney, adrenal,
spleen, heart, lungs and gonads of male and female animals treated
with Acetylsalicylic Acid-L-Threonine Ester or reference drug
(Aspirin) groups did not show any histopathological changes and
were found to be normal and comparable to that of animals of
vehicle control group. However few animals treated with reference
drug (Aspirin) showed mild fatty changes in the cardiac muscle
fibers of heart and mild catarrhal changes of gastric mucosa. Human
Clinical Trials:
[0531] Several blood clotting time studies were performed on a
limited number of human volunteers with Acetylsalicylic
Acid-L-Threonine Ester. Results are shown in FIGS. 8-11. Attention
is directed to FIG. 8 which compares the average clotting time
after 325 mg and 81 mg Acetylsalicyclic Acid L-Threonine Ester and
Bayer Aspirin is administered to human volunteers. The first set of
columns in FIG. 8 labeled "Normal" is the average clotting time
observed prior to the adminstration of the Aspirin or its threonine
ester derivative thereof.
[0532] As clearly shown by the data in FIG. 8, Acetylsalicylic
Acid-L-Threonine Ester at low dose of 80 mg was as effective as 325
mg Bayer Aspirin (with baseline correction), and obviously more
effective than Bayer 81 mg Aspirin.
[0533] The data are tabulated in Table 29. The data on which the
graphs of FIG. 8 are based are summarized in Table 29 below:
TABLE-US-00033 TABLE 29 AVERAGE CLOTTING TIME (MIN) 325 mg 81 mg 81
mg ASA-T 325 mg ASA-T 81 mg ASA-T ESTER ASA Bayer ESTER ASA Bayer
ESTER Volunteer 1 Volunteer 2 Volunteer 1 Volunteer 2 Volunteer 3
Normal 4.5 4.5 4 4 4 With ASA-T ESTER 6.2 6.0 5.2 NA 5.1 With ASA
(Bayer) 5.7 5.7 NA 4.5 NA
[0534] FIG. 10.0 depicts the percentage increase in clotting time
of Acetylsalicylic Acid-L-Threonine Ester relative to Aspirin
(Bayer) at 81 mg dose based on 5-day average increase. This plot
was derived from the data in Table 29. The two Acetylsalicylic
Acid-L-Threonine Ester blocks shown above correspond to two
separate volunteers who took the test drug over a period of 5 days.
The third volunteer took Bayer Aspirin for 5 days. As per FIG. 2,
increase in clotting time occurred on the very first day of drug
intake, and remained higher than Bayer ASA during the subsequent
administrations.
[0535] Pharmacokinetic Results in 4 volunteers who took
Acetylsalicylic Acid-L-Threonine Ester versus two volunteers who
took Bayer Aspirin at 325 mg dose are shown in FIG. 11.
[0536] FIG. 11 is a plot of the concentration of Aspirin versus
time in 4 volunteers who took Acetylsalicylic Acid-L-Threonine
Ester and 2 volunteers who took Bayer Aspirin. No Aspirin was found
in blood for volunteers who took Acetylsalicylic Acid-L-Threonine
Ester. The two volunteers who took Bayer Aspirin showed Plasma
levels of Aspirin.
[0537] This is shown in FIG. 12 which depicts the concentration of
salicyclic Acid in human plasma with respect to four volunteers who
took Acetylsalicyclic Acid-L-Threonine Ester and two volunteers who
took Aspirin.
[0538] Rapid influx and efflux of Salicylic Acid from Aspirin
administration was seen in the two volunteers who took Aspirin
(FIG. 12). There was prolonged and sustained formation and
disappearance of Salicylic acid in volunteers who took
Acetylsalicylic Acid-L-Threonine Ester (FIG. 7). Without wishing to
be bound, this phenomenon is believed to be attributed to the high
likelihood of Acetylsalicylic Acid-L-Threonine Ester exhibiting
sustained, specific and irreversible acetylation of the platelets
in the portal circulation. Since there was no aspirin found in the
systemic circulation after oral dosing of Acetylsalicylic
Acid-L-Threonine Ester, it is likely that site specific action of
acetylation of the platelets has been achieved, and the unwanted
effect of aspirin in the endothelial system has been avoided. Thus
it is evident from the human pharmacokinetic studies, clinical
trials, and rat gastric mucosa irritation results, Acetylsalicylic
Acid-L-Threonine Ester is a superior anti-platelet drug that does
not have the toxicity of Aspirin and which exhibits a much better
Therapeutic Index.
[0539] Therefore, Acetylsalicylic Acid-L-Threonine Ester is a
Superior Anti platelet drug than acetylsalicyclic acid, for several
reasons: [0540] 1. It does not produce any gastric irritation;
[0541] 2. It does not inhibit prostaglandin synthesis; [0542] 3. It
does not have any COX-1 or COX-2 activity on the endothelial or
vascular tissues, as no ASA from Acetylsalicylic Acid-L-Threonine
Ester reaches systemic circulation. [0543] 4. Thus, none of the
side effects of Aspirin is observed with Acetylsalicylic
Acid-L-Threonine Ester.
[0544] There are a number of screening tests to determine the
utility of the derivatives created according to the disclosed
methods. These include both in vitro and in vivo screening
methods.
[0545] The in vitro methods include acid/base hydrolysis of the
derivatives, hydrolysis in pig pancreas hydrolysis in rat
intestinal fluid, hydrolysis in human gastric fluid, hydrolysis in
human intestinal fluid, and hydrolysis in human blood plasma. These
assays are described in Simmons, D M, Chandran, V R and Portmann, G
A, Danazol Amino Acid Derivatives: In Vitro and In Situ
Biopharmaceutical Evaluation, Drug Development and Industrial
Pharmacy, Vol 21, Issue 6, Page 687, 1995, the contents of all of
which are incorporated by reference.
[0546] The NSAID amino acid derivatives of the present invention
are effective in treating diseases or conditions in which NSAIDs
normally are used. These amino acid derivatives disclosed herein
enhance the therapeutic benefits of the NSAIDs by reducing or
eliminating biopharmaceutical and pharmacokenetic barriers
associated with each of them. However it should be noted that these
amino acid derivatives themselves will have sufficient activity
without releasing any active drug in the mammals. Since the amino
derivatives are more soluble in water than Ibuprofen or other
NSAIDs, they do not need to be associated with a carrier vehicle,
such as alcohol or castor oil which may be toxic or produce
unwanted side reactions. Moreover, oral formulations containing the
NSAID derivatives are absorbed into the blood and are quite
effective.
[0547] Thus, the amino acid derivatives of the present invention,
discussed hereinabove enhance the therapeutic benefits by removing
biopharmaceutical and pharmacokenetic barriers of existing
drugs.
[0548] Furthermore, these amino acid derivatives are easily
synthesized in high yields using reagents which are readily and
commercially available.
IV. Proline Derivative of Acetaminophen
Overview:
[0549] The procedure for the synthesis of the L-proline ester of
acetaminophen is outlined in Synthetic Sequence section. The
synthesis is exemplary. The complete procedure and analytical data
is given in the Experimental Section. Acetaminophen (10 g) was
coupled with Boc-L-proline with EDC in the presence of a catalytic
amount of DMAP. Once the reaction was complete (3 hours at room
temperature), the solution was washed with water. After drying over
sodium sulfate, filtration, and concentration the crude protected
amino acid ester of acetaminophen was purified by flash
chromatography on silica gel. The procedure generated the protected
L-proline ester of acetaminophen in 72%. The protecting group was
removed by dissolving the ester in dichloromethane and passing
hydrogen chloride through the solution at room temperature. After
filtration, the final salt was stirred in tetrahydrofuran until
pure. The yield for the deprotection step was 91.4% after
filtration and drying under high vacuum at 90.degree. C. for 4
hours. Synthetic Sequence: ##STR59## Synthesis of the L-proline
Ester of Acetaminophen: a) EDC, DMAP, CH.sub.2Cl.sub.2; b) HCl (g),
CH.sub.2Cl.sub.2. Experiment Section:
[0550] The synthesis of SPI0014 was conducted in one batch.
Reagents mentioned in the experimental section were purchased at
the highest obtainable purity from Lancaster, Sigma-Aldrich, or
Acros, except for solvents, which were purchased from either Fisher
Scientific or Mallinkrodt.
SPI0014: Pyrrolidine-2(S)-carboxylic Acid 4-acetylamino-phenyl
ester, Hydrochloride
[0551] A mixture of Boc-L-proline (14.39 g, 68.80 mmole),
acetaminophen (10.02 g, 66.28 mmole), EDC (12.9 g, 67.29 mmole) and
DMAP (1.10 g, 9.0 mmole) in anhydrous dichloromethane (100 mL) was
stirred for 3 hours at room temperature under an argon atmosphere.
After 3 hours, water (120 mL) was added. After mixing for 5
minutes, the layers were separated and the dichloromethane fraction
was washed with water (120 mL) and dried over sodium sulfate (5 g).
After filtration, concentration under reduced pressure, and drying
under high vacuum, the remaining oil (24.10 g) was purified by
flash chromatography on silica gel (100 g, 0.035-0.070 mm, 6 nm
pore diameter), eluting with hexanes/ethyl acetate (1:2). After
concentration of the product containing fractions under reduced
pressure and drying at high vacuum until the weight was constant,
the experiment produced the protected acetaminophen-L-proline ester
SPI001401 (16.71 g, 72.3% yield) as a white solid (foam).
##STR60##
[0552] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=8.83 (1/2H, s),
8.70 (1/2H, s), 7.58 (1/2H, d, J=7.5 Hz), 7.46 (1/2H, d, J=7.5 Hz),
6.96 (2H, m), 4.47 (1H, m), 3.59-3.45 (2H, m), 2.36 (1H, m),
2.17-1.90 (6H, m), 1.46 (9H, m).
[0553] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=171.91, 171.75,
169.02, 154.44, 153.78, 146.36, 146.21, 121.44, 121.23, 120.82,
80.41, 80.17, 59.16, 46.78, 46.55, 31.06, 30.11, 28.50, 24.57,
24.28, 23.78.
[0554] The protected acetaminophen-L-proline ester SPI001401 (16.60
g, 47.64 mmole) was dissolved in dichloromethane (400 mL) and
hydrogen chloride gas was passed through the solution for 2 hours
at room temperature. The remaining solids were allowed to settle
(for 1 hour). The dichloromethane was carefully decanted away from
the white precipitate. Tetrahydrofuran (200 mL) was added to the
precipitate and the mixture stirred for 2 hours under an argon
atmosphere. After filtration, the remaining white solid was dried
under high vacuum at 90.degree. C. until the product weight was
constant (4 hours). The experiment produced acetaminophen-L-proline
ester, hydrochloride SPI0014 (12.4 g, 91.4% yield) as a white
solid.
[0555] .sup.1H NMR (300 MHz, CDCL.sub.3-DMSO): .delta.=10.41 (1H,
br s), 10.26 (1H, s), 9.55 (1H, br s), 7.70 (2H, d, J=9 Hz), 7.12
(2H, d, J=9 Hz), 4.66 (t, 1H, J=8.4 Hz), 3.33 (2H, m), 2.43 (1H,
m), 2.28 (1H, m), 2.08 (s, 3H), 2.04 (2H, m).
[0556] .sup.13C NMR (75 MHz, CDCL.sub.3-DMSO): .delta.=168.08,
167.25, 144.55, 137.40, 121.12, 119.64, 58.53, 45.33, 27.74, 23.86,
23.08.
[0557] HPLC analysis:
[0558] 99.45% purity; rt=5.733 min; Luna C18 5 u column (sn
167917-13); 4.6.times.250 mm; 254 nm; 15% MeOH/85% hexane sulfonate
buffer (110 mMol, pH=6); 35 C; 20 ul inj.; 1 ml/min; 5 mg/mL sample
size.
[0559] CHN analysis:
[0560] calc.: C, 54.84; H, 6.02; N, 9.84. found: C, 54.66; H, 5.98;
N, 9.65.
[0561] Melting point: 221-222.degree. C.
V. Amino Acid Derivative of Cyclosporine A
[0562] The macrocyclic immunosuppresants comprise a class of
structurally distinctive, cyclic, poly, N-methylated undecaptides,
and similar semi-synthetic macrolide structures commonly possessing
pharmacological, in particular immunosuppressive, anti-inflammatory
and/or anti-parasitic activity. The first of the cyclosporine to be
isolated was the naturally occurring fungal metabolite Ciclosporin
or Cyclosporine also known as cyclosporine A, which has the
formula: ##STR61## wherein MeBmt represents
N-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L) threonyl residue of the
formula ##STR62## in which -x-y- is CH.dbd.CH-- (trans). Other
similar products include, sirolimus (b), tacrolimus (c), and
pimecrolimus (d), having the following structures: ##STR63##
[0563] The class comprised by the cyclosporines is thus now very
large indeed and includes, for example, [Thr].sup.2-, [Val].sup.2-,
[Nva].sub.2- and [Nva].sup.2-[Nva].sup.5-Ciclosporin (also known as
cyclosporines C, D, G and M respectively),
[Dihydrop-MeBmt].sup.1-[Val].sup.2-ciclosporin (also known as
dihydro-cyclosporine D), [(D)Ser].sup.8-Ciclosporin,
[MeIle].sup.11-Ciclosporin, [(D)MeVal].sup.11-Ciclosporin (also
known as cyclosporine H), [MeAla].sup.6-Ciclosporin,
[(D)Pro].sup.3-Ciclosporin and so on.
[0564] In accordance with conventional nomenclature for
cyclosporines, these are defined throughout the present
specification and claims by reference to the structure of
cyclosporine (i.e., Cyclosporine A). This is done by first
indicating the amino acid residues present which differ from those
present in cyclosporine (e.g., "[(D)Pro].sup.3" to indicate that
the cyclosporine in question has a -(D)Pro- rather than -Sar-
residue at the 3-position) and then applying the term Cyclosporine
to characterize remaining residues which are identical to those
present in Cyclosporine A.
[0565] As used herein, the term "cyclosporines" refers to the
various types of cyclosporines, in which x-y in the MeBmt residue
has a cis or trans CH.dbd.CH or in which x-y therein is also
included in those derivatives in which one or more of those amino
acids in positions 2-11 of Cyclosporine A is replaced by a
different amino acid. It is preferred, however, that not more than
two of the amino acids are replaced in the formula of cyclosporine
A and more preferentially not more than one of the amino acids is
replaced by an amino acid.
[0566] In addition, amino acid residues referred to by
abbreviation, e.g., -Ala-, -MeVal- and -.alpha.Abu-, are, in
accordance with conventional practice, to be understood as having
the (L)-configuration unless otherwise indicated, e.g. as in the
case of "-(D)Ala-". Residue abbreviations preceded by "Me" as in
the case of "-MeLeu-", represent .alpha.-N-methylated residues.
Individual residues of the cyclosporine molecule are numbered, as
in the art, clockwise and starting with the residue -MeBmt-,
dihydro-MeBmt- etc. . . . in position 1. The same numerical
sequence is employed throughout the present specification and
claims.
[0567] Because of their unique pharmaceutical potential, the
macrocyclic immunosuppressants have attracted considerable
attention in the press. The term "macrocyclic immuno-suppressants"
includes various natural and semi-synthetic derivatives of
cyclosporine, and other macrolides such as sirolimus, tacrolimus
and pimecrolimus. The primary area of clinical investigation for
the above drugs has been as immunosuppressive agents, in particular
in relation to its application to recipients of organ transplants,
e.g., heart, lung, combined heart-lung, liver, kidney, pancreatic,
bone-marrow, skin and corneal transplants, and in particular
allogenic organ transplants. These drugs are also used in the
treatment of psoriasis, atompic dermatitis, rheumatoid arthritis
and nephritic syndrome.
[0568] Macrocyclic immunosuppressants are also useful for treating
various autoimmune diseases and inflammatory conditions and
especially inflammatory conditions with an aetiology, including an
autoimmune component, such as arthritis (for example, rheumatoid
arthritis, arthritis chronica progredient and arthritis deformons)
and rheumatic diseases. Specific autoimmune diseases for which
cyclosporine therapy has been proposed or applied include,
autoimmune hematological disorder (including, e.g., hemolytic
anemia, aplastic anemia, pure red cell anemia, and idiopathic
thrombocytopaenia), systemic lupus erythematosus, polychondritis,
sclerodoma, Wegener granulamatosis, dermatomyositis, chronic active
hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome,
idiopathic sprue, autoimmune inflammatory bowel disease, including,
e.g., ulcerative colitis and Crohn's disease), endocrine
opthalmopathy Graves disease, sarcoidosis, multiple sclerosis,
primary billiary cirrhosis, juvenile diabetes (diabetes mellitus
type I), uvetis (anterior and posterior), keratoconjunctivitis
sicca and vernal keratoconjunctivitis, interstial lung fibrosis,
psoriatic arthritis, atopic dermatitis and glomerulonephritis (with
and without nephrotic syndrome, e.g., including idiopathic
nephritic syndrome or minimal change nephropathy).
[0569] Furthermore, macrocyclic immunosuppressants also have
applicability as an anti-parasitic, in particular anti-protozoal
agent, and are suggested to be useful for treating malaria,
coccidiomycosis and schistomsomiasis. More recently, they have been
taught to be useful as agents for reversing or abrogating
anti-neoplastic agent resistance contumors, and the like.
[0570] Despite the very major contribution which macrocyclic
immunosuppressants have made, difficulties have been encountered in
providing more effective and convenient means of administration
(e.g., galenic formulations, for example, oral dosage form, which
are both convenient and for the patient as well as providing
appropriate bioavailability and allowing dosaging at an appropriate
and controlled dosage rate) as well as the reported occurrence of
undesirable side reactions; in particular nephrotoxic reactions
have been obvious serious impediments to its wider use or
application.
[0571] Moreover, the above mentioned macrocyclic immunosuppressants
are characteristically highly hydrophobic and readily precipitate
in the presence of even very minor amounts of water, e.g., on
contact with the body (e.g., stomach fluids). It is accordingly
extremely difficult to provide e.g., oral formulations, which are
acceptable to the patient in terms of form and taste, which are
stable on storage and which can be administered on a regular basis
to provide suitable and controlling patient dosaging.
[0572] Proposed liquid formulations, e.g., for oral administration
of macrocyclic immunosuppressants, have heretofore been based
primarily on the use of ethanol and oils or similar excipients as
carrier media. Thus, the commercially available macrocyclic
immunosupressant drink-solution employs ethanol and olive oil or
corn-oil as carrier medium in conjunction with solvent systems
comprising e.g., ethanol and LABRIFIL and equivalent excipients as
carrier media. Thus, the commercially available macrocyclic
immunosupressant drink solution employs ethanol and olive oil or
corn-oil as carrier medium in conjunctions with a Labrifil as a
surfactant. See e.g., U.S. Pat. No. 4,388,307. Use of the drink
solution and similar composition as proposed in the art is,
however, accompanied by a variety of difficulties.
[0573] Further, the palatability of the known oil based system has
proved problematic. The taste of the known drink-solution is, in
particular, unpleasant. Admixture with an appropriate flavored
drink, for example, chocolate drink preparation, at high dilution
immediately prior to ingestion has generally been practiced in
order to make regular therapy at all acceptable. Adoption of oil
based systems has also required the use of high ethanol
concentrations to itself inherently undesirable, in particular
where administration to children is forseen. In addition,
evaporation of the ethanol, e.g., from capsules (adopted in large
part, to meet problems of palatability, as discussed or other forms
(e.g., when opened)) results in the development of a macrocyclic
immunosupressant precipitate. When such compositions are presented
in, for example, soft gelatin encapsulated form an additional
problem arises. This particular difficulty necessitates packaging
of the encapsulated product in an air-tight component, for example,
an air-tight blister or aluminum-foil blister package. This in turn
renders the product both bulky and more expensive to produce. The
storage characteristics of the aforesaid formulations are, in
addition, far from ideal.
[0574] Bioavailability levels achieved using existing oral
macrocyclic immunosupressant dosage system are also low and exhibit
wide variation between individuals, individual patient types and
even for single individuals at different times during the course of
therapy. Reports in the literature indicate that currently
available therapy employing the commercially available macrocyclic
immunosupressant drink solution provides an average absolute
bioavailability of approximately 30% only, with the marked
variation between individual groups, e.g., between liver
(relatively low bioavailability) and bone-marrow (relatively high
bioavailability) transplant recipients. Reported variation in
bioavailability between subjects has varied from one or a few
percent for some patients, to as much as 90% or more for others.
And as already noted, marked change in bioavailability for
individuals with time is frequently observed. Thus, there is a need
for a more uniform and high bioavailability of macrocyclic
immunosupressant in patients.
[0575] Use of such dosage forms is also characterized by extreme
variation in required patient dosaging. To achieve effective
immunosuppressive therapy, blood or blood serum levels compounds of
the cyclosporin have to be maintained within a specified range.
This required range can in turn, vary, depending on the particular
condition being treated, e.g., whether therapy is to prevent
transplant rejection or for the control of an autoimmune disease,
or condition and on whether or not alternative immunosuppressive
therapy is employed concomitantly with any of the
immunosuppressants of the formula described herein. Because of the
wide variations in bioavailability levels achieved with
conventional dosage forms, daily dosages needed to achieve required
blood serum levels will also vary considerably from individual to
individual and even for a single individual. For this reason it is
necessary to monitor blood/blood-serum levels of patients receiving
macrocyclic immunosuppressant therapy at regular and frequent
intervals. Monitoring of blood/blood-serum levels, which is
generally performed by RIA or equivalent immunoassay technique,
e.g. employing monoclonal antibody based technology, has to be
carried out on a regular basis. This is inevitably time consuming
and inconvenient and adds substantially to the overall cost of
therapy.
[0576] It is also the case that blood/blood serum macrocyclic
immunosuppressant levels achieved using available dosage systems
exhibit extreme variation between peak and trough levels. That is,
for each patient, effective macrocyclic immunosuppressant levels in
the blood vary widely between administrations of individual
dosages.
[0577] There is also a need for providing macrocyclic
immunosuppressant in a water soluble form for injection. It is well
known that Cremephore L used in a current formulations of
macrocyclic immunosuppressants is a polyoxyethylated derivative of
castor oil and is a toxic vehicle. There have been a number of
incidences of anaphylaxis due to the castor oil component. At
present there is no formulation that would allow the macrocyclic
immunosuppressants to be in aqueous solution at the concentrations
needed due to poor water solubility of the drug.
[0578] Beyond all these very evident practical difficulties lies
the occurrence of undesirable side reactions already alluded to,
observed employing available oral dosage forms.
[0579] Several proposals to meet these various problems have been
suggested in the art, including both solid and liquid oral dosage
forms. An overriding difficulty which has, however, remained is the
inherent insolubility of the macrocyclic immunosuppressants in
aqueous media, hence preventing the use of a dosage form which can
contain macrocyclic immunosuppressants in sufficiently high
concentration to permit convenient use and yet meet the required
criteria in terms of bioavailability, e.g. enabling effective
resorption from the stomach or gut lumen and achievement of
consistent and appropriately high blood/blood-serum levels.
[0580] The particular difficulties encountered in relation to oral
dosaging with macrocyclic immunosuppressants have inevitably led to
restrictions in the use of macrocyclic immunosuppressant therapy
for the treatment of both relatively less severe or endangering
disease conditions. A particular area of difficulty in this respect
has been the adoption of macrocyclic immunosuppressant therapy in
the treatment of autoimmune diseases and other conditions affecting
the skin, for example, for the treatment of atopic dermatitis and
psoriasis and, as also widely proposed in the art, for hair growth
stimulation, e.g. in the treatment of alopecia due to ageing or
disease.
[0581] Thus while oral macrocyclic immunosuppressant therapy has
shown that the drug is of considerable potential benefit to
patients suffering e.g. from psoriasis, the risk of side-reaction
following oral therapy has prevented common use. Various proposals
have been made in the art for application of macrocyclic
immunosuppressants, e.g. cyclosporine, in topical form and a number
of topical delivery systems have been described. Attempts at
topical application have however failed to provide any demonstrably
effective therapy.
[0582] However, the present invention overcomes the problems
described hereinabove. More specifically, an embodiment of the
present invention is an amino acid derivative of macrocyclic
immunosuppressant which significantly enhances its solubility in
aqueous solutions, thereby avoiding the need to utilize a carrier,
such as ethanol or castor oil when administered as a solution.
Moreover, the amino acid derivatives of macrocyclic
immunosuppressants, in accordance with the present invention, do
not exhibit the side effects of the prior art formulations.
Further, the inventor has found that the macrocyclic
immunosuppressant amino acid derivatives of the present invention
enhance their absorption when administered in the form of the amino
acid derivative to a patient, thereby enhancing significantly their
bioavailability and efficacy.
[0583] Accordingly, in one aspect, the present invention is
directed to an amino acid derivative of macrocyclic
immunosuppressants. The amino acid derivative consists of an amino
acid esterified to the free hydroxy group present on the
macrocyclic immunosuppressents; e.g., on the side chain of
cyclosporine, sirolimus, tacrolimus and either one of the hydroxyl
groups of the pimecrolimus molecule.
[0584] For example, an aspect of the present invention is directed
to, the compounds of the formulas ##STR64## ##STR65## or
pharmaceutically acceptable salts thereof; wherein CYCLO represents
the residues at positions 2-11 of the cyclosporine molecule; x-y is
CH.dbd.CH or CH.sub.2CH.sub.2 and AA is an amino acid or a
dipeptide of the formula GLY-AA. In the latter case, GLY is glycine
and AA is any .alpha.-amino acid but preferably the L-.alpha.-amino
acid and more preferably the naturally occurring amino acids,
especially in the L-form. In the dipeptide structure, an AA is
attached to the drug via OH group using glycine as the spacer.
Glycine is esterified to cyclosporine and then glycine is bonded to
any AA via amide linkage using amino group of glycine and
carboxylic acid group of AA.
[0585] The present invention is also directed to a pharmaceutical
composition comprising a therapeutically effective amount of the
compounds of the Formulae a-d above and a pharmaceutical carrier
therefor.
[0586] In another embodiment, the present invention is directed to
a method of treating a patient in need of macrocyclic
immunosuppressant therapy, which method comprises administering to
said patient an effective amount of the compounds of Formulae
a-d.
[0587] In a further embodiment, the present invention is directed
to a method of enhancing the solubility of a macrocyclic
immunosuppressant in an aqueous solution comprising reacting said
immunosuspressant having a hydroxy group thereof, e.g., hydroxy
functionality in the MeBmt moiety at position 1 of the cyclosporine
molecule as well as the specified hydroxyl functions in formulas
b-d, with an amino acid or acylating derivative thereof under ester
forming conditions, and isolating the product or by using a simple
amino acid or a dipeptide structure or acylating derivative wherein
the AA is attached to drug using glycine as the spacer and
isolating and isolating the product thereof.
[0588] In a still further embodiment, the present invention is
directed to a method of enhancing the bioavailability of a
macrocyclic immunosuppressant when administered to a patient which
comprises reacting the immunosuspressant having a hydroxy group
thereon, e.g., the hydroxy functionality in the MeBmt moiety in
position 1 of the cyclosporine molecule, with an amino acid or
acylating derivative under ester forming conditions and as well as
the specified hydroxyl functions in formulas b-d with an amino acid
or acylating derivative thereof under ester forming conditions or
by using a simple amino acid or a dipeptide structure wherein the
AA is attached to the drug using glycine as the spacer and
isolating the product thereof and administering said product to the
patient.
Overview:
[0589] The procedure for the synthesis of the N-(L-proline)-glycine
and N-(L-lysine)-glycine esters of Cyclosporine A is outlined in
Synthetic Sequence section. These examples are exemplary of the
synthetic scheme using amino acids. The complete procedure and
analytical data is given in the Experimental Section. Cyclosporine
A (15 g) was coupled with chloroacetic anhydride (4 equivalent) in
anhydrous pyridine. The experiment produced the chloroacetate ester
of Cyclosporine A (SPI001201, 14 g, 88% yield) in good yield. The
chloroacetate ester (10.1 g) was then treated with sodium azide in
DMF to generate the azidoacetate ester of Cyclosporine A
(SPI001202, 9.9 g, 97% yield). The azidoacetate (9.8 g) was then
reduced with tin chloride (9 g) to prepare the glycine ester of
Cyclosporine A (8.54 g, 89% yield). The glycine ester of
Cyclosporine A (SPI001203) was then coupled with a two-fold excess
of either boc-L-proline or Boc-L-lysine using EDC as the coupling
agent. After purification by column chromatography, the boc
protecting groups were removed from the dipeptide esters of
Cyclosporine A at low temperature (5.degree. C.) by treatment with
2M hydrochloric acid in diethyl ether. The L-lysine-glycine ester
salt of Cyclosporine A did not require additional purification and
was dried. The L-proline-glycine ester salt of Cyclosporine A
required purification. The salt was converted to the free-base with
sodium bicarbonate and purified by filtration through silica gel
(eluting with acetone). The salt was then formed at low temperature
with dilute anhydrous hydrochloric acid and dried. Synthetic
Sequence: ##STR66## ##STR67## Synthesis of the
N-(L-proline)-glycine and N-(L-Lysine)-glycine Esters of
Cyclosporine A: a) Pyridine; b) NaN.sub.3, DMF; c) SnCl.sub.2,
Methanol; d) boc-L-lysine, EDC; e) boc-L-proline, EDC; f) HCl,
Et.sub.2O. Experimental Section:
[0590] The synthesis of SPI0022 and SPI0023 was conducted in
batches. Generally a small-scale experiment was performed first
followed by a larger batch. Reagents mentioned in the experimental
section were purchased at the highest obtainable purity from
Aldrich, Acros, or Bachem, except for solvents, which were
purchased from either Fisher Scientific or Mallinckrodt. The
Cyclosporine A (USP grade) used in these procedures was provided by
Signature Pharmaceuticals, Inc. ##STR68##
[0591] Cyclosporine A (15.01 g, 0.0124 moles) was dissolved in
anhydrous pyridine (35 mL) at room temperature, under an argon
atmosphere. The solution was cooled to 5.degree. C. in an ice/water
batch and chloroacetic anhydride (9.10 g, 0.053 moles) was added.
After stirring for 10 minutes, the ice bath was removed and the
solution was allowed to stir under an argon atmosphere at room
temperature for 17 hours. After 17 hours, diethyl ether (200 mL)
was added. The ether was washed with water (2.times.100 mL) and
dried for 1 hour over sodium sulfate (10 g). After filtration and
concentration under reduced pressure, the remaining yellow foam was
dried under high vacuum (1 hour at room temperature) and purified
by flash chromatography on silica gel (200 g), eluting with
heptane/acetone (2:1). After combining and concentrating the
product containing fractions, the remaining light yellow foam (14.8
g) was purified a final time by crystallization from hot diethyl
ether (140 mL). After cooling (-10.degree. C., 2 hours),
filtration, and drying under high vacuum, the procedure generated
the chloroacetate ester of Cyclosporine A SPI001201 as a white
solid (14.0 g, 88.3% yield).
Cyclosporine A Chloroacetate Ester:
[0592] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=8.50 (d, 1H,
J=9.6 Hz), 7.95 (d, 1H, J=6.6 Hz), 7.46 (d, 1H, J=9.0 Hz), 7.40 (d,
1H, J=7.8 Hz), 5.35-4.52 (m, 15H), 4.37 (t, 1H, J=7.2 Hz), 4.12 (d,
1H, J=14.7 Hz), 3.89 (d, 1H, J=14.7 Hz), 3.45-3.0 (m, 15H), 2.8-2.5
(m, 6H), 2.5-1.5 (m, 16H), 1.5-0.7 (m, 53H).
[0593] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=173.78, 173.37,
172.86, 172.61, 171.28, 171.18, 170.91, 170.79, 168.78, 167.64,
167.18, 128.77, 126.68, 75.46, 65.95, 58.89, 57.47, 55.80, 55.31,
54.86, 54.34, 50.19, 48.91, 48.35, 48.02, 44.80, 40.96, 39.44,
37.07, 35.93, 33.85, 33.25, 32.40, 31.74, 31.50, 30.38, 30.12,
29.82, 29.53, 25.13, 24.92, 24.78, 24.40, 23.99, 23.75, 22.85,
21.94, 21.41, 21.25, 20.84, 19.85, 18.79, 18.32, 17.89, 17.82,
15.46, 15.24, 10.08. ##STR69##
[0594] The chloroacetate ester of Cyclosporine A SPI001201 (10.10
g, 7.89 mmole) was dissolved in anhydrous N,N-dimethlformamide (30
mL) at room temperature. Sodium azide (2.15 g, 33.0 mmole) was
added. The mixture was allowed to stir at room temperature for 24
hours in the dark, under an argon atmosphere. After 24 hours,
diethyl ether (150 mL) was added and the precipitate was filtered.
The ether was washed with water (2.times.00 mL), dried over sodium
sulfate (15 g) for 30 minutes, filtered, and concentrated under
reduced pressure. The remaining white solid was dried under high
vacuum for 1 hour at room temperature. The experiment produced the
azidoacetate ester of Cyclosporine A SPI001202 (9.90 g, 97% yield)
as a white solid, which was used without further purification.
Cyclosporine A Azidoacetate Ester:
[0595] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=8.48 (d, 1H,
J=9.3 Hz), 7.95 (d, 1H, J=6.9 Hz), 7.45 (d, 1H, J=9.0 Hz), 7.39 (d,
1H, J=7.8 Hz), 5.5-4.5 (m, 15H), 4.31 (t, 1H, J=6.6 Hz), 4.04 (d,
1H, J=17.3 Hz), 3.53 (d, 1H, J=17.3 Hz), 3.45-3.0 (m, 15H), 2.8-2.5
(m, 6H), 2.5-1.5 (m, 16H), 1.5-0.7 (m, 53H).
[0596] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=173.76, 173.32,
172.82, 172.53, 171.13, 170.89, 170.76, 170.69, 169.70, 168.20,
167.49, 128.63, 126.61, 74.96, 58.91, 57.39, 55.56, 55.21, 54.80,
54.23, 50.14, 48.99, 48.23, 48.24, 47.93, 44.71, 40.89, 39.33,
39.22, 37.02, 35.83, 33.81, 32.96, 32.31, 31.67, 31.42, 30.31,
30.09, 29.76, 29.47, 25.08, 24.92, 24.84, 24.67, 24.51, 24.40,
23.94, 23.82, 23.71, 21.85, 21.33, 21.25, 20.82, 19.79, 18.71,
18.25, 17.92, 17.81, 15.17, 10.03. ##STR70##
[0597] The azidoacetate ester of Cyclosporine A SPI001202 (9.80 g,
7.62 mmole) was dissolved in methanol (250 mL) at room temperature.
Water (40 mL) was added followed by tin (II) chloride (5 g, 26.3
mmole). The solution was allowed to stir for 1 hour at room
temperature when an additional quantity of tin (II) chloride (4 g,
21.0 mmole) was added. The solution was allowed to stir for an
additional 2 hours at room temperature. Water (200 mL) containing
ammonium hydroxide (40 mL, 29%) was added. After filtration, the
solution was concentrated (to 200 mL) under reduced pressure. The
remaining aqueous solution was extracted with ethyl acetate
(2.times.200 mL). The ethyl acetate fractions were combined, dried
over sodium sulfate (20 g), filtered and concentrated under reduced
pressure. The remaining clear foam was purified by filtration
through silica gel (150 g), eluting with dichloromethane/methanol
(20:1). The procedure generated the glycine ester of Cyclosporine A
as a clear, solid foam (8.54 g, 89% yield).
Glycine Ester of Cyclosporine A:
[0598] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=8.60 (d, 1H,
J=9.6 Hz), 8.06 (d, 1H, J=6.9 Hz), 7.53 (d, 1H, J=8.4 Hz), 7.51 (d,
1H, J=6.6 Hz), 5.7-4.52 (m, 15H), 4.41 (t, 1H, J=6.9 Hz), 3.5-3.0
(m, 17H), 2.82-2.5 (m, 8H), 2.5-1.5 (m, 16H), 1.5-0.7 (m, 53H).
[0599] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=174.10, 173.67,
173.23, 172.72, 172.55, 171.18, 171.10, 170.73, 170.61, 169.68,
167.77, 128.82, 126.42, 73.83, 58.57, 57.32, 55.99, 55.20, 54.74,
54.31, 50.08, 48.82, 48.28, 47.90, 44.70, 43.81, 40.74, 39.33,
39.24, 37.02, 35.84, 33.72, 33.07, 32.39, 31.72, 31.41, 30.25,
29.98, 29.74, 29.51, 25.05, 24.81, 24.73, 24.54, 24.31, 23.91,
23.78, 23.68, 21.86, 21.33, 21.25, 20.68, 19.76, 18.74, 18.24,
17.94, 17.79, 15.18, 10.03. ##STR71##
[0600] The glycine ester of Cyclosporine A (SPI001203, 2.0 g, 1.59
mmole) was dissolved in anhydrous dichloromethane (25 mL) with
boc-L-lysine (1.31 g, 3.78 mmole) and EDC (0.75 g, 3.9 mmole),
under an argon atmosphere at room temperature. The boc-L-lysine was
prepared from the dicyclohexylamine salt (2.0 g in 50 mL ether) by
extraction with cold potassium hydrogen sulfate solution (1 g in 50
mL water) followed by cold water (2.times.50 mL). The ether
containing the boc-L-lysine was dried over sodium sulfate (5 g),
filtered, concentrated and dried under high vacuum for one hour at
room temperature. A few crystals of DMAP were added to the mixture
of EDC, boc-L-lysine, and the glycine ester of Cyclosporine A and
the solution was allowed to stir for 4 hours at room temperature.
The dichloromethane solution was extracted with DIUF water (50 mL),
5% sodium bicarbonate solution (50 mL), and with DIUF water (50
mL). After drying over sodium sulfate (10 g), the dichloromethane
solution was filtered and concentrated under reduced pressure. The
remaining white foam (3.01 g) was purified by flash column
chromatography on silica gel (50 g), eluting with heptane/acetone
(2:1). The product containing fractions were combined, concentrated
under reduced pressure, and dried under high vacuum. The purified
protected intermediate (2.34 g white solid, 92.8% yield) was placed
in a flask under an argon atmosphere, which was cooled in an
ice-water bath. Cold anhydrous 2 M hydrochloric acid in diethyl
ether (20 mL) was added and the solution stirred for 8 hours (at
5.degree. C.). The mixture was slowly allowed to warm to room
temperature overnight. After stirring for a total of 20 hours, the
flask was cooled again in an ice-water bath for 30 minutes. The
product was filtered and dried under high vacuum for 1 hour at room
temperature and then at 50.degree. C. for 4 hours. The experiment
produced Cyclosporine A N-(L-lysine)-glycine ester, dihydrochloride
trihydrate (SPI0022, 1.59 g, 73.9% yield) as a white solid.
[0601] .sup.1H NMR (300 MHz, CDCl.sub.3, NMR data is for the free
base): .delta.=8.58 (d, 1H, J=9.3 Hz), 8.04 (d, 1H, J=6 Hz), 7.80
(d, 1H, J=6 Hz), 7.49 (d, 2H, J=8.4 Hz), 5.70-4.6 (m, 17H), 4.41
(m, 1H), 4.28 (dd, 1H, J=17, 7.2 Hz), 3.67 (d, 1H, J=17 Hz), 3.46
(s 3H), 3.4-2.8 (m, 16H), 2.8-2.5 (m, 8H), 2.5-1.35 (m, 24H),
1.5-0.7 (m, 50H).
[0602] .sup.13C NMR (75 MHz, CDCl.sub.3, NMR data is for the free
base): .delta.=175.23, 173.77, 173.34, 172.75, 172.63, 171.34,
171.22, 170.94, 170.84, 170.91, 169.89, 169.70, 128.74, 126.67,
74.41, 58.82, 57.43, 55.91, 55.21, 54.81, 54.42, 50.17, 48.89,
48.31, 47.98, 44.78, 41.92, 40.82, 40.69, 39.44, 39.32, 27.19,
35.91, 34.88, 33.71, 33.25, 33.12, 32.44, 31.83, 31.50, 30.38,
30.06, 29.81, 29.55, 25.14, 24.90, 24.52, 24.43, 24.00, 23.76,
21.93, 21.42, 21.29, 20.81, 19.84, 18.82, 18.32, 17.96, 17.86,
15.21, 10.10.
[0603] CHN analysis:
[0604] Calculated for
C.sub.70H.sub.128Cl.sub.2N.sub.14O.sub.15.3H.sub.2O: C, 55.50; H,
8.92; and N, 12.74. found: C, 58.28; H, 8.98; and N, 13.16.
[0605] HPLC analysis:
[0606] 99.60% purity; r.t.=14.763 min.; 80% acetonitrile/20% Tris
base in DIUF water; 1 mL/min; 60 C; Synergi Hydro RP, 4 u column
(serial # 163383-7), 4.6.times.250 mm; 20 ul; UV=210 nm.
[0607] Melting point: 196.0-198.degree. C. (uncorrected)
##STR72##
[0608] The glycine ester of Cyclosporine A (SPI001203, 7.50 g, 5.95
mmole) was dissolved in anhydrous dichloromethane (50 mL) with
boc-L-proline (2.56 g, 11.90 mmole) and EDC (2.28 g, 11.9 mmole),
under an argon atmosphere at room temperature. A few crystals of
DMAP were added to the mixture of EDC, boc-L-proline, and the
glycine ester of Cyclosporine A and the solution was allowed to
stir for 3 hours at room temperature. The dichloromethane solution
was extracted with DIUF water (50 mL), 5% sodium bicarbonate
solution (2.times.50 mL), and with DIUF water (50 mL). After drying
over sodium sulfate (10 g), the dichloromethane was filtered and
concentrated under reduced pressure. The remaining white foam (9.50
g) was purified by flash column chromatography on silica gel (150
g), eluting with heptane/acetone (2:1 followed by 1:1). The product
containing fractions were combined, concentrated under reduced
pressure, and dried under high vacuum (7.94 g white solid, 91.7%
yield) for 10 minutes at room temperature.
[0609] The purified protected intermediate (6.46 g) was placed in a
flask under an argon atmosphere, which was cooled in an ice-water
bath. Cold anhydrous 2 M hydrochloric acid in diethyl ether (150
mL) was added and the solution stirred for 8 hours (at 5.degree.
C.). The mixture was slowly allowed to warm to room temperature
overnight. After stirring for a total of 20 hours, the flask was
cooled again in an ice-water bath for 30 minutes. The product was
filtered and dried under high vacuum for 30 minutes at room
temperature. The Cyclosporine A N-(L-proline)-glycine ester,
hydrochloride (5.17 g, 84.6% yield, and 90% purity by HPLC) was
converted to the free base by dissolving the salt in DIUF water (25
mL) that contained sodium bicarbonate (1 g). The free base was
extracted with dichloromethane (3.times.25 mL), which was dried
over sodium sulfate (5 g), filtered and concentrated. The remaining
off-white solid (5 g) was purified by filtration through silica gel
(100 g), eluting with acetone. The product containing fractions
were combined, concentrated under reduced pressure, and dried under
high vacuum for 30 minutes at room temperature. The hydrochloride
salt was regenerated by dissolving the free base (3.8 g) in diethyl
ether (25 mL) and adding it to anhydrous 2M hydrochloric acid (5
mL) in heptane (50 mL), while cooling in an ice-water bath. After
20 minutes at 5.degree. C., the white solid was filtered and dried
under high vacuum for 6 hours at room temperature. The experiment
produced Cyclosporine A N-(L-proline)-glycine ester, hydrochloride
(SPI0023, 3.8 g) as a white solid.
[0610] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=14.20 (br s, 2H),
8.62 (d, 1H, J=10 Hz), 8.06 (d, 1H, J=6.9 Hz), 7.61 (d, 1H, J=8.1
Hz), 7.48 (d, 1H, J=9 Hz), 5.70-5.50 (m, 3H), 5.40-4.60 (m, 12H),
4.37 (m, 1H), 4.20 (d, 1H, J=18 Hz), 3.97 (d, 1H, J=18 Hz), 3.70
(m, 1H), 3.45 (s, 3H), 3.23-3.08 (m, 12H), 2.66 (s, 3H), 2.60 (s,
3H), 2.50-1.80 (m, 15H), 1.78-1.20 (m, 15H), 1.15-0.66 (m,
46H).
[0611] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=174.15, 173.49,
172.67, 172.59, 171.86, 171.20, 171.13, 171.02, 170.83, 169.68,
168.77, 167.55, 128.30, 127.10, 80.09, 75.58, 62.65, 59.35, 57.36,
55.53, 55.30, 54.78, 54.35, 53.60, 50.25, 50.09, 48.92, 48.18,
48.12, 44.62, 40.59, 40.02, 39.43, 39.30, 37.13, 35.88, 33.74,
33.07, 32.19, 32.01, 31.86, 31.50, 31.43, 30.43, 29.93, 29.72,
29.30, 29.16, 27.56, 26.04, 25.00, 24.86, 24.74, 24.39, 20.96,
19.81, 18.71, 18.26, 18.09, 17.85, 17.79, 15.09, 14.30, 10.00.
CHN analysis:
[0612] Calculated for C.sub.69H.sub.122ClN.sub.13O.sub.14: C,
59.48; H, 8.83; and N, 13.07. found: C, 59.84; H, 9.02; and N,
12.65.
[0613] HPLC analysis:
[0614] 99.59% purity; r.t.=10.613 min.; 85% acetonitrile/15% Tris
base in DIUF water; 1.2 mL/min; 60 C; Synergi Hydro RP, 4 u column
(serial # 163383-7), 4.6.times.250 mm; 20 ul; UV=210 nm.
[0615] Melting point: 197.0-199.degree. C. (uncorrected)
[0616] The amino acid derivatives of cyclosporin of the present
invention are effective in treating diseases or conditions in which
macrocyclic immunosuppressants normally are used. These derivatives
are transformed within the body to release the active compound and
enhances the therapeutic benefits of the macrocyclic
immunosuppressants by reducing or eliminating biopharmaceutical and
pharmacokenetic barriers associated with each of them. However it
should be noted that these derivatives themselves will have
sufficient activity without releasing any active drug in the
mammals. Since the derivatives are more soluble in water then
cyclosporine or other macrocyclic immunosuppressants, it does not
need to be associated with a carrier vehicle, such as alcohol or
castor oil which may be toxic or produce unwanted side reactions.
Moreover, oral formulations containing the derivatives of the
derivatives are absorbed into the blood and are quite
effective.
[0617] Thus, the derivative of cyclosporin of the present invention
enhances the therapeutic benefits by removing biopharmaceutical and
pharmacokenetic barriers of existing drugs.
[0618] Furthermore, the derivatives are easily synthesized in high
yields using reagents which are readily and commercially
available.
Animal Effiacy Study Results for Cyclosporine Derivatives:
[0619] A simple method to test the efficacy of cyclosporine A and
its analogs, Cyclosporine-glycine-proline (Cyclosporine GP) ester,
Cyclosporine-glycine-lysine (Cyclosporine GL) ester and
Cyclosporine-glycine (Cyclosporine G) ester is to treat ulcerative
colitis induced by administering 5% Dextran Sodium Sulfate (DSS) ad
libitum to laboratory mice. This model has been used consistently
with most immunosuppressants to demonstrate both actual treatment
after induction of colitis, and also prophylactic treatments.
Results from both types of studies are shown below.
Calculation of Disease Activity Index (DAI)
[0620] Percent animal body weight loss, stool consistency and
occult blood at the end of the study will be taken into
consideration to calculate Disease Activity Index ("DAI") of each
animal. The following scoring system outlined in Table 30 was for
the percent body weight loss, stool consistency and occult blood:
TABLE-US-00034 TABLE 30 Score Weight loss (%) Stool consistency
Blood in feces 0 0 or gain Normal Negative 1 1-4.9 Soft +/- 2
5.0-9.9 Mixed (soft & + Diarrhea) 3 10-15 Diarrhea ++ 4 >15
Bloody Diarrhea +++ (Gross blood)
The scores for each parameter for each mouse at the end of study
are added together and divided by three (number of observations) to
determine DAI. The mean group DAI is the determined. A score of 3
was assigned for any animal that died during the testing. Results
from the Study are Indicated in Table 31:
[0621] In this study, DSS administration is coupled with
administration of either test or reference drug from day 1. No of
mice used were 10 in each group: TABLE-US-00035 TABLE 31 Drug Mean
DAI Vehicle 1.500 Cyclosporine A (Ref) 1.400 Cyclosporine-G 1.531
Cyclosporine-GL 1.100 Cyclosporine-GP 1.062
[0622] While Cyclosporine G didn't perform as expected in this
model, Cyclosporine GP had statistically significant performance
compared to Cyclosporine A.
Results from Disease Treatment Study:
[0623] Much more dramatic results were obtained when the mice were
initially treated with DSS to induce colitis. Once clear symptoms
of colitis were seen, DSS was replaced by drinking water, and
treatment started with test and reference drugs, including vehicle
control. All results are mean of 10 mice. The mean DAI os tabulated
hereinbelow in Table 32: TABLE-US-00036 TABLE 32 Drug Mean DAI
Vehicle 1.832 Cyclosporine A (Ref) 0.300 Cyclosporine-G 0.100
Cyclosporine-GL 0.532 Cyclosporine-GP 0.066
[0624] Clearly Cyclosporine G and Cyclosporine-GP showed
statistically significant results, significant improvement over
Cyclosporine reference drug. Notice in the treatment study, 5 out
of 10 mice died in the vehicle control, 1 died with cyclosporine-GL
treatment, and none died in the cyclosporine-A, Cyclosporine-G and
Cyclosporine-GP treated groups.
Development of Cyclosporine GP Ophthalmic Preparation:
[0625] Clear, aquous solutions of the Cyclosporine GP was prepared
by dissolving sufficient quantities of Cyclosporine GP in distlled
water, and pH adjusted to around 5 to produce 0.2% w/v
concentrations. Allowable quantities of HCl, and alcohol as per US
FDA ophthalmic additive content regulations were added to keep the
mixture stable and solubilized in the ophthalmic preparation. The
resulting clear solution was stable for 3 months at room
temperature. This solution appeared pharmaceutically elegant than
currently available formulations such as Restasis.RTM. and other
cloudy preparations using cremaphor or castor oil to make emulsion
of cyclosporine in water.
[0626] As indicated hereinabove the solubility of the amino acid
derivatives of the macrocyclic immunosuppressent with an OH group
functionality group thereon with which the amino acid can form an
ester linkage is significantly enhanced in aqueous solution
relative to the corresponding macroscopic immunosuppressents which
are not linked to the amino acids. For example, the solubility of
cyclosporine in water is approximately 30 microgram/ml (US patent
publication 20040138108). The solubility of
Cyclosporine-glycine-proline ester in aquous solution at room
temperature was 60 times that of Cyclosporine A in water, and
equaled approximately 2000 micrograms/ml.
VI. Valproic Acid Esters
[0627] Valproic acid (2-Propylpentanoic acid) is low molecular
weight carboxylic acid derivative which is widely used as an
anti-convulsive agent, useful in the treatment of epilepsy and also
possess vasodilatation activity in the brain to relieve migraine
headaches. It is administered orally to control epileptic episodes
in humans and also alleviate severe pain associated with migraine
headaches. ##STR73##
[0628] Valproic acid has been shown to have a large number of
therapeutic applications, which are quite varying and somewhat
surprising. For example, in addition to its efficacy in the
treatment of epilepsy and migraine headaches, it has been shown to
be effective in the treatment of certain psychiatric illnesses,
such as bipolar disorder, mood stabilization, control of
aggression, impulsivity in personality disorder, agitation in
dementia, and has also been of use as adjunct therapy in the
treatment of post traumatic stress disorder (PTSD).
Mechanism of Action:
[0629] In spite of being used in the treatment of epilepsy for a
number of years, the exact mechanism of action of Valproic acid is
still unknown. It has been postulated that it exerts its action by
increasing concentration of gamma-amino butyric acid (GABA) in the
brain. Gamma-amino butyric acid is a neurotransmitter, a chemical
that nerves use to communicate with one another.
[0630] Valproate is the drug of choice in myoclonic epilepsy, with
or without generalized tonic-clonic seizures, including juvenile
myoclonic epilepsy of Janz that begins in adolescence or early
adulthood. Photosensitive myoclonus is usually easily controlled.
Valproate also is effective in the treatment of benign myoclonic
epilepsy, postanoxic myoclonus, and, with clonazepam, in severe
progressive myoclonic epilepsy that is characterized by
tonic-clonic seizures as well. It also may be preferred in certain
stimulus-sensitive (reflex, startle) epilepsies.
[0631] Although Valproate may be effective for infantile spasms, it
is relatively contraindicated in children whose spasms are due to
hyperglycinemia or other underlying metabolic (mitochondrial)
abnormalities. In general, atonic and akinetic seizures in patients
with Lennox-Gastaut syndrome are difficult to control, but
Valproate is the drug of choice for treatment of mixed seizure
types. Since this drug has been useful in some patients who are
refractory to all other antiepileptic drugs, it may warrant a trial
in nearly all nonresponsive patients regardless of seizure
type.
[0632] In spite of it usefulness, hepatotoxicity may be fatal, but
is idiosyncratic and not preventable by routinely monitoring liver
enzymes. Hepatotoxicity occurs in very young children, most often
those on multiple anticonvulsants. Valproate-induced cytopenias may
be dose-related and warrant monitoring of complete blood counts
during therapy. Encephalopathy with hyperammonemia without liver
function test abnormalities may occur. Pregnant women in their
first month are at risk for neural tube defects.
[0633] Valproic acid is a low molecular weight liquid with
characteristic odor. Taken orally, it has unpleasant taste and can
severely irritate mouth and throat. In order to convert Valproic
acid into a solid dosage form convenient for oral administration, a
number of derivatives with covalent and ionic bond with the
carboxylic acid have been made. A simple sodium salt of Valproic
acid, resulting in Valproate sodium is available as a solid.
However a stable coordination complex, know as Divalproex sodium
was formed by partial neutralization of two molecules of Valproic
acid with one atom of sodium. This product is the most widely
available commercial Valproic acid hemisalt. It is marketed by
Abbott Laboratories in the USA under the brand name Depakote.RTM..
Depakote.RTM. is also available in extended release formulation for
oral administration.
[0634] A significant disadvantage of Valproic acid is that in
liquid form, it is difficult to administer. Furthermore,
administration of Valproic acid in different forms does not
uniformly produce desired bioavailability. For example, the overall
bioavailability of Valproate from Valproic acid, its sodium salt,
Divalproex.RTM., and their extended release formulations are not
quite interchangeable. Since continuous monitoring of the plasma
profile of a patient to whom Valproic acid is administered is
essential, any change in plasma concentration due to changes in the
formulation can adversely affect the overall therapeutic
outcome.
[0635] The amino acid derivatives of Valproic acid improve the
therapeutic effectiveness, uniform blood profile, develop
pharmaceutically elegant formulation and reduce first pass
metabolism, exhibited by valproic acid alone.
[0636] Until now there has been no pharmaceutical preparation
available in the market that can deliver Valproic acid without
harmful side effects. The present invention, however, has produced
a number of water soluble, non-toxic derivatives of Valproic acid
which are suitable for delivering Valproic acid consistently in the
body without any harmful side effects and without the needs for
expensive additives and excipients.
[0637] Accordingly, in one aspect, the present invention is
directed to a class of amino acid derivatives of Valproic acid or
acylating derivative thereof. The derivative consists of the
hydroxyl group of an amino acid esterified to the free carboxyl
group present on the Valproic acid molecules. In another
embodiment, the amine group of the amino acid is reacted with COOH
of the valproic acid or acylating derivative thereof to form an
amide linkage.
[0638] More specifically, an embodiment of the present invention is
directed to, the compounds of the formula ##STR74## or
pharmaceutically acceptable salts thereof wherein R is either NH-AA
or O-AA and AA is an amino acid less an amino group in NHAA or less
an hydroxy group in O-AA, in which either an amine group or the
hydroxyl group is reacted with the carboxylic acid group of
Valproic Acid.
[0639] Recently A small proof of concept study (Depletion of latent
HIV-1 infection in vivo: a proof-of-concept study Lehrman G, Hogue
I B, Palmer S, Jennings C, Spina C A, Wiegand A, Landay A L, Coombs
R W, Richman D D, Mellors J W, Coffin J M, Bosch R J, Margolis D M.
The Lancet--Vol. 366, Issue 9485, 13 Aug. 2005, Pages 549-555)
demonstrated that Valproic Acid might reduce the pool of dormant
HIV infected cells in the body.
[0640] As part of its life cycle, HIV needs to insert its genetic
material into the DNA of the human CD4 T-cells which it infects.
These incorporated genes of the virus are then used as template for
the production of new virus particles in active cells. This process
leads to the cells being over-activated, eventually causing most of
them to commit suicide or `apoptosis` and the CD4 cell counts to
fall.
[0641] However, a small proportion of the HIV-infected cells do not
die, but become dormant within the lymph node or other areas of the
body. Since they are not actively producing new HIV particles, the
genes of the virus remain hidden within the nuclei of these cells.
These cells, which are distributed throughout the body, are highly
stable and long-lasting, but remain ready to start pumping out new
HIV particles if they become activated by stimulation of the immune
system.
[0642] Currently available anti-retroviral drug combinations (known
as AIDS cocktail) can reduce HIV to undetectable levels in the
blood, but they can only do so by preventing HIV from making copies
of itself, or `replicating` in the body. Anti-retroviral drugs
typically can not remove HIV's genes that are integrated into the
human cells' DNA, leaving the reservoir of the HIV in latent
infected cells untouched.
[0643] Valproic Acid (VPA) which is currently used worldwide for
the treatment of bipolar disorders and epilepsy is an inhibitor of
a cellular enzyme called histone deacetylase 1 (HDAC1). This enzyme
has been shown to be crucial in keeping HIV's gene hidden within
the host cells' DNA.
[0644] In the test tube, studies have shown that VPA can stimulate
the release of the HIV from resting infected T-Cells. Consequently,
scientists are trying to find out whether giving the drug to
HIV-positive patients could stimulate the release of resting cells
to start producing and expressing HIV particles and thus eliminate
the hidden stores of HIV from the body. Once the viral particles
are exposed to anti-retroviral drugs, the disease is cured since
there are no hidden reservoirs in the body in the short run.
Problems with VPA Therapy:
[0645] 1. A small proof of concept study explained above was done
in a limited number of patients (4 patients), and it was not a
proof of efficacy study. When these 4 patients were treated for 18
weeks with Valproic acid ("VPA"), the investigators saw a decline
in resting cells containing HIV genes between 68 to 84%, and in the
4.sup.th patient, had smaller reduction to 29%.
2. VPA is not without its severe side effects and adverse
reactions. FDA has mandated boxed bold letter warnings for this
drug to be included in all product inserts. Serious box warning
include hepatotoxicity, teratogenicity, pancreatitis.
3. Other serious side effects include urea cycle disorders (UCD),
somnolence in the elderly, thrombocytopenia, hyperammonemia, birth
defects, mania, and various GI, Nervous system, respiratory system,
skin disorders and weight gain.
4. While VPA has shown to be carcinogenic and mutagenic in animal
models, such effects in humans are unknown.
5. Dr. Robert Siliciano from the Johns Hopkins University is of the
opinion that "its extremely unlikely that this approach would
work", who is one of the scientists who discovered dormant
infection of the HIV in the mid-1990s.
[0646] 6. According to Dr. Siliciano, 99.9999% of the hidden HIV
infection has to be "kicked" out of the dormant human CD4 T-cells.
In the proof of concept study, it was shown that reduction levels
varied between 29 to 84%, far short of the 99.9999% demanded by Dr.
Siliciano.
Solution to the Problem:
[0647] The amino acid derivatives of valproic acid represent the
solution to the problem. They are effective in expelling the virus,
e.g., HIV virus from the human immune cell reservoirs.
[0648] Table 33 lists as examples, 6 amino acid derivatives of
valproic acid effective in expelling the virus: TABLE-US-00037
TABLE 33 No Drug Mol. Wt 1 Valproic Acid L-Threonine Ester 281.78 2
Valproic Acid Serine Ester 267.76 3 Valproic Acid Serine Amide
Nitrate 276.29 4 Valproic Acid Tyrosine Ester 343.85 5 Valproic
Acid Hydroxyproline Amide 257.33 6 Valproic Acid Hydroxyproline
Ester 257.33
[0649] The present invention is also directed to a pharmaceutical
composition comprising a therapeutically effective amount of the an
amino acid derivative of Valproic acid and a pharmaceutical carrier
therefor.
[0650] In another embodiment, the present invention is directed to
a method of treating a patient in need of Valproic acid therapy,
which method comprises administering to said patient an effective
amount of the amino acid derivative of the Valproic acid.
[0651] In a further embodiment, the present invention is directed
to a method of converting liquid Valproic acid into a solid powder
by reacting the carboxyl functionality of the Valproic acid with
either the amine or hydroxyl functionality of an amino acid or
acylating derivative thereof and isolating the product(s)
thereof.
[0652] In a still further embodiment, the present invention is
directed to a method of substantially and in a therapeutically
efficacious manner, reducing or eliminating the potential first
pass metabolism thereby improving the consistent therapeutic effect
by administering to a patient an amino acid derivative which
comprises reacting the COOH functionality of the Valproic acid
molecule with either the NH.sub.2 or OH functionality of the amino
acids to form an ester or amide covalent bond, respectively, and
isolating the product thereof and administering said product to the
patient.
[0653] The present inventor has found that when naturally occurring
amino acids are esterified to Valproic acid, the resulting
derivatives are pharmaceutically elegant free flowing powders, and
are rapidly absorbed into the body and release non-toxic amino
acids upon cleavage in the body and require none of the
emulsifiers, additives and other excipients associated with
volproic acid.
[0654] Furthermore, the amino acid derivatives of Valiproic acid
are effective anti-epileptics and exhibit such effect intact. Thus
the current amino acid derivatives are effective anti-epileptics
and useful in the treatment of a number of psychiatric illnesses
and exhibit such potential with or without releasing the active
parent drug.
[0655] The amino acid derivative of Valproic acid exhibits a bulk
density which is much higher than the corresponding sodium salts,
and they are suitable for compacting large weight tablets and
capsules. Furthermore, the amino acid derivatives of Valproic acid
do not exhibit bitter taste and unusual odor of the Valproic
acid.
[0656] As indicated hereinabove, the amino acid derivatives are
effective anti-epileptics with or without releasing Valproic acid.
However, when administered in vivo, the amino acid derivative
release in vivo the active drug with all its pharmacological and
psychoactive properties.
[0657] In conclusion, the amino acid derivative of Valproic acid
clearly provides a number of advantages over Valproic acid. For
example, they are less toxic than the corresponding valproic acid
when administered in vivo, the valproic acid amino acid derivatives
may be metabolized to cleave the amino acid moiety from the
valproic acid, which are non-toxic. This results in a high
therapeutic index. Secondly, the amino acid derivatives may or may
not cleaved in the body to release Valproic acid. The amino acid
derivative of valproic acid exhibits the same utility as valproic
acid itself. Furthermore, due to their high water solubility, they
can be easily administered by either forming an in-situ solution
just before IV administration using lyophilized sterile powder or
providing the drug in solution in prefilled syringe or bottles for
infusion. The amino acid esters are more stable than Valproic acid
since the COOH group in Valproic acid is blocked so as to minimize
any reaction with bases. Thus the amino acid derivatives of
Valproic acid are more effective than Valproic acid itself without
the toxicity and other pharmaceutical problems associated with
current marketed formulations of valproic acid.
[0658] The procedures for the synthesis of the L-serine,
L-threonine, and L-hydroxyproline esters of valproic acid
(2-propylpentanoic acid) are outlined in Synthetic Sequence section
and is exemplary for the preparation of the various derivatives of
the present invention. The complete procedure and analytical data
is given in the Experimental Section. In general, valproic acid
(2-8 g, in batches) was coupled with the N-benzyloxy/benzyl ester
protected amino acids using EDC in the presence of a catalytic
amount of DMAP. Once the reactions were complete (20 hours at room
temperature), the mixture was extracted with DIUF water, dried over
sodium sulfate, and concentrated under reduced pressure. The crude
material was either used directly for the deprotection step or
purified by column chromatography. The procedure generated the
protected amino acid esters of valproic acid in yields ranging from
72% to 92%. The protecting groups were removed by hydrogenation (30
psi H.sub.2) in the presence of 10% palladium on carbon. The amino
acid esters of valproic acid were extracted away from the palladium
catalyst with ethanol, concentrated, and dried. The final salts
were formed by acidification with hydrochloric acid. The crude
salts (yields ranging from 57% to 92%) were then purified by the
methods described in the Experimental Section. Synthetic Sequence:
##STR75## ##STR76## Synthesis of the L-serine, L-threonine, and
L-hydroxyproline Esters of Valproic Acid: a) EDC, DMAP,
CH.sub.2Cl.sub.2; b) H.sub.2, 10% Pd/C, EtOAc; c) HCl. Experimental
Section:
[0659] The synthesis of SPIC001, SPIC002 and SPIC003 were conducted
in one or two batches. Reagents mentioned in the experimental
section were purchased at the highest obtainable purity from
Lancaster, Sigma-Aldrich, Acros, or Bachem, except for solvents,
which were purchased from either Fisher Scientific or
Mallinkrodt.
1) SPIC001: 2-Propylpentanoic Acid 2(S)-amino-2-carboxy-ethyl
ester, Hydrochloride
L-Serine-valproic Acid Ester, Hydrochloride
[0660] A mixture of 2-propylpentanoic acid (valproic acid, 6.48 g,
44.93 mmole), N-carbobenzyloxy-L-serine benzyl ester (Z-Ser-OBzl,
14.80 g, 44.93 mmole), EDC (8.61 g, 44.91 mmole), and DMAP (549 mg,
4.49 mmole) in anhydrous dichloromethane (50 mL) was stirred under
an argon atmosphere at room temperature for 20 hours. After 20
hours, the dichloromethane was washed with water (3.times.50 mL),
dried over magnesium sulfate (5 g), filtered and concentrated under
reduced pressure. The remaining colorless oil (20.87 g) was
purified by column chromatography on silica gel (150 g, 0.035-0.070
mm, 6 nm pore diameter), eluting with hexanes/ethyl acetate (3:1).
After concentration of the product containing fractions under
reduced pressure and drying under high vacuum until the weight was
constant, the experiment produced the protected L-serine-valproate
ester SPIC00101 (18.9 g, 92% yield) as a colorless oil.
##STR77##
[0661] .sup.1H NMR (300 MHz, DMSO): .delta.=7.96 (1H, d, J=8.1 Hz),
7.35 (10H, m), 5.14 (2H, s), 5.05 (2H, s), 4.51 (1H, m), 4.29 (2H,
m), 2.29 (1H, m), 1.50-1.25 (4H, m), 1.25-1.10 (4H, m), 0.80 (6H,
t, J=6.6 Hz).
[0662] .sup.13C NMR (75 MHz, DMSO): .delta.=174.88, 169.15, 155.85,
136.58, 135.45, 128.26, 128.18, 127.47, 127.71, 127.57, 66.32,
65.66, 62.47, 53.09, 44.20, 33.86, 33.79, 19.95, 13.85.
[0663] The protected L-serine-valproate ester SPIC00101 (18.9 g,
41.48 mmole) was dissolved in ethanol (60 mL) and ethyl acetate (60
mL) at room temperature and added to a Parr bottle (500 mL) that
contained 10% palladium on carbon (3.0 g, 50% wet) under a nitrogen
atmosphere. The nitrogen atmosphere was replaced with hydrogen gas
(30 psi). After 4 hours of shaking, additional palladium catalyst
(1.0 g) in ethanol\ethyl acetate (1:1, 100 mL) was added and the
reaction mixture shook overnight under hydrogen gas (30 psi) at
room temperature. After 24 hours the catalyst was removed by
filtration through a thin layer of activated carbon. The ethanol
and ethyl acetate were concentrated under reduced pressure at room
temperature. After drying under high vacuum, the remaining solids
were acidified with hydrochloric acid in diethyl ether (2M, 24.6
mL). The mixture was stored in a refrigerator for two hours before
filtration and washing with additional cold diethyl ether (10 mL).
After filtration, the remaining white solid was dried at room
temperature under high vacuum until the product weight was constant
(24 hours). The experiment produced L-serine-valproic acid ester,
hydrochloride SPIC001 (6.34 g, 57% yield) as a white solid.
##STR78##
[0664] .sup.1H NMR (300 MHz, DMSO): .delta.=8.73 (br s, 3H), 4.47
(dd, 1H, J=12.9, 4.5 Hz), 4.31 (dd, 2H, J=12.9, 3.6 Hz), 2.36 (m,
1H), 1.50 (m, 2H), 1.39 (m, 2H), 1.20 (m, 4H), 0.84 (t, 6H, J=7
Hz).
[0665] .sup.13C NMR (75 MHz, DMSO): .delta.=174.67, 168.19, 61.84,
51.16, 44.12, 33.76, 33.58, 20.07, 19.92, 13.97, 13.89.
[0666] HPLC analysis:
[0667] 98.49% purity; rt=4.767 min; Luna C18 5 u column (sn
167917-13); 4.6.times.250 mm; 254 nm; 33% ACN/66% DIUF water; 35 C;
20 ul inj.; 1 ml/min; 20 mg/mL sample size; sample dissolved in
mobile phase.
[0668] CHN analysis:
[0669] calc.: C, 49.34; H, 8.28; N, 5.23. found: C, 49.22; H, 8.35;
N, 5.24.
[0670] Melting point: 159-160.degree. C.
2) SPIC002:
4(R)-(2-Propyl-pentanoyloxy)-pyrrolidine-2(S)-carboxylic Acid
(L-Hydroxyproline-valproic Acid Ester)
[0671] A mixture of 2-propylpentanoic acid (valproic acid, 4.32 g,
30 mmole), N-carbobenzyloxy-L-hydroxyproline benzyl ester
(Z-Hyp-OBzl, 10.66 g, 30 mmole), EDC (5.74 g, 30 mmole), and DMAP
(366 mg, 3 mmole) in anhydrous dichloromethane (30 mL) was stirred
under an argon atmosphere at room temperature for 20 hours. After
20 hours, the dichloromethane was washed with water (3.times.30
mL), dried over magnesium sulfate (5 g), filtered and concentrated
under reduced pressure. The remaining colorless oil SPIC00201
(11.95 g, 24.7 mmole, 82.4% yield) was used without purification.
##STR79##
[0672] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.29 (10H, m),
5.28-5.00 (5H, m), 4.55 (1/2H, t, J=8 Hz), 4.46 (1/2H, t, J=8 Hz),
3.80-3.60 (2H, m), 2.43-2.16 (3H, m), 1.60-1.45 (2H, m), 1.40-1.32
(2H, m), 1.28-1.20 (4H, m), 0.86 (6H, m).
[0673] .sup.13C NMR (75 MHz, DMSO): .delta.=174.74, 171.40, 171.05,
153.79, 153.31, 136.34, 136.20, 135.57, 135.38, 128.24, 128.13,
127.95, 127.87, 127.67, 127.52, 127.28, 127.10, 72.29, 71.53,
66.34, 66.10, 57.66, 57.19, 52.27, 51.89, 44.13, 40.33, 35.78,
34.79, 34.04, 33.92, 33.35, 20.00, 19.91, 13.79, 13.73.
[0674] The protected L-hydroxyproline-valproate ester SPIC00201
(17.24 g, 35.79 mmole) was dissolved in ethanol (50 mL) and ethyl
acetate (100 mL) at room temperature and added to a Parr bottle
(500 mL) that contained 10% palladium on carbon (3.5 g, 50% wet)
under a nitrogen atmosphere. The nitrogen atmosphere was replaced
with hydrogen gas (30 psi). After 15 hours of shaking, the catalyst
was removed by filtration through a thin layer of celite and
activated carbon. The ethanol and ethyl acetate mixture was
concentrated under reduced pressure at room temperature. After
drying overnight under high vacuum at room temperature, the
experiment produced L-hydroxyproline-valproic acid ester SPIC002
(9.2 g, 99.8% yield) as a white solid. In order to remove trace
impurities, the zwitterion was purified by reverse-phase column
chromatography (50 g ODS silica gel) in two batches. The zwitterion
was placed on the column in DIUF water and eluted with mixture of
DIUF water/methanol (2:1, 1:1, 1:2, 100% methanol).
[0675] The product containing fractions were combined, concentrated
under reduced pressure at 20.degree. C. (or less), and dried under
high vacuum at room temperature until the weight was constant (24
hours, 6.4 g white solid recovered). ##STR80##
[0676] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=12.40 (br s, 1H),
8.32 (br s, 1H), 5.28 (m, 1H), 4.11 (t, 1H, J=7.2 Hz), 3.59 (m,
1H), 3.34 (br d, 1H, J=10.5 Hz), 2.50-2.22 (m, 3H), 1.62-1.50 (m,
2H), 1.50-1.32 (m, 2H), 1.32-1.19 (m, 4H), 0.88 (t, 6H, J=7.2
Hz).
[0677] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=175.99, 173.35,
71.83, 59.56, 49.77, 45.08, 36.19, 34.51, 20.87, 14.31.
[0678] HPLC analysis:
[0679] 99.20% purity; r.t.=7.228 min.; 70% DIUF water/30%
acetonitrile; 1 mL/min; 36.8 C; Luna C18, 5 u column (serial #
167917-13), 4.6.times.250 mm; 22 ul injection; sample dissolved in
mobile phase.
[0680] CHN analysis:
[0681] calc.: C, 60.68; H, 9.01; N, 5.44. found: C, 60.58; H, 9.12;
N, 5.48. Melting point: 179.0-180.0.degree. C.
3) SPIC003: 2-Propyl-pentanoic Acid
2(S)-amino-2-carboxy-1(R)-methyl-ethyl Ester, Hydrochloride
L-Threonine-valproic Acid Ester, Hydrochloride
[0682] A mixture of 2-propylpentanoic acid (valproic acid, 4.32 g,
30 mmole), N-carbobenzyloxy-L-threonine benzyl ester (Z-Thr-OBzl,
10.30 g, 30 mmole), EDC (5.74 g, 30 mmole), and DMAP (366 mg, 3.0
mmole) in anhydrous dichloromethane (30 mL) was stirred under an
argon atmosphere at room temperature for 20 hours. After 20 hours,
the dichloromethane was washed with water (3.times.30 mL), dried
over magnesium sulfate (5 g), filtered and concentrated under
reduced pressure. The remaining colorless oil (13.44 g) was
purified by column chromatography on silica gel (100 g, 0.035-0.070
mm, 6 nm pore diameter), eluting with hexanes/ethyl acetate (4:1).
After concentration of the product containing fractions under
reduced pressure and drying under high vacuum until the weight was
constant, the experiment produced the protected
L-threonine-valproate ester SPIC00301 (12.65 g, 89.8% yield) as a
colorless oil. ##STR81##
[0683] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.40-7.05 (11H,
m), 5.45 (1H, m), 5.17-5.02 (4H, m), 4.53 (1H, d, J=9.6 Hz), 2.24
(1H, m), 1.58-1.40 (2H, m), 1.40-1.15 (9H, m), 0.86 (6H, m).
[0684] .sup.13C NMR (75 MHz, DMSO): .delta.=174.24, 169.29, 156.48,
136.61, 135.34, 128.26, 128.20, 127.74, 127.67, 127.58, 69.04,
66.33, 65.78, 57.62, 44.50, 33.89, 33.80, 20.03, 19.91, 16.40,
13.87.
[0685] The protected L-threonine-valproate ester SPIC00301 (12.65
g, 26.9 mmole) was dissolved in ethanol (50 mL) and ethyl acetate
(50 mL) at room temperature and added to a Parr bottle (500 mL)
that contained 10% palladium on carbon (2.53 g, 50% wet) under a
nitrogen atmosphere. The nitrogen atmosphere was replaced with
hydrogen gas (30 psi). After 20 hours the catalyst was removed by
filtration through a thin layer of activated carbon, washing with
ethanol (25 mL). The ethanol and ethyl acetate were concentrated
under reduced pressure at room temperature. After drying under high
vacuum, the remaining solids (6.13 g) were acidified with
hydrochloric acid (3.1 mL conc.) in DIUF water (50 mL). The
solution was filtered a second time through activated carbon and
dried overnight in a freeze-dryer. The experiment produced
L-threonine-valproic acid ester, hydrochloride SPIC003 (6.52 g,
86.0% yield) as a white solid.
[0686] The combined batches of the L-threonine-valproic acid ester,
hydrochloride SPIC003 (8.8 g) were purified by crystallization form
acetonitrile. After the salt was dissolved in hot acetonitrile (225
mL), the material was treated activated acrbon, filtered, and
placed in a 5.degree. C. refrigerator overnight. The white solids
were filtered after 18 hours, washed with cold acetonitrile (10
mL), and dried under high vacuum at room temperature until the
product weight was constant (24 hours). The process recovered
L-threonine-valproic acid ester, hydrochloride SPIC003 (6.82 g,
77.5% recovery) as a white solid. ##STR82##
[0687] .sup.1H NMR (300 MHz, DMSO): .delta.=8.71 (br s, 3H), 5.28
(m, 1H), 4.16 (d, 1H, J=2.7 Hz), 2.33 (m, 1H), 1.56-1.40 (m, 2H),
1.37-1.27 (m, 5H), 1.21-1.13 (m, 4H), 0.84 (t, 6H, J=6.6 Hz).
[0688] .sup.13C NMR (75 MHz, DMSO): .delta.=173.97, 168.19, 67.69,
55.42, 44.43, 33.95, 33.78, 20.07, 19.95, 16.54, 13.94.
[0689] HPLC analysis:
[0690] 98.88% purity; r.t.=4.864 min.; 70% DIUF water/30%
acetonitrile; 1 mL/min; 40 C; Luna C18, 5 u column (serial #
211739-42), 4.6.times.250 mm; 20 ul injection; sample dissolved in
mobile phase.
[0691] CHN analysis:
[0692] calc.: C, 51.15; H, 8.59; N, 4.97. found: C, 51.29; H, 8.59;
N, 4.98.
[0693] Melting point: 144.degree. C.
[0694] The procedure for the synthesis of the L-serine,
L-serine(O-nitroxy ester), L-hydroxyproline, and L-tyrosine
conjugates of valproic acid (2-propylpentanoic acid) is outlined in
Synthetic Sequence section. The complete procedure and analytical
data is given in the Experimental Section. In general, valproic
acid (2-8 g, in batches) was coupled with the corresponding
protected amino acids. The protected intermediates were purified by
column chromatography and the protective groups were removed. The
final amides were purified by crystallization from ether. A portion
of the L-serine amide was nitrated at low temperature with slight
excess of a standard nitrating solution to form the nitroxy ester,
which could be separated from starting material by crystallization
from toluene. The final products were dried to a constant weight
before analysis by NMR, HPLC, melting point, and CHN analysis.
Synthetic Sequence: ##STR83## ##STR84## Synthesis of the L-serine,
L-tyrosine, and L-hydroxyproline Conjugates of Valproic Acid: a)
Et.sub.3N; b) LiOH; c) EDC, DMAP; d) H.sub.2, 10% Pd/c, HCl; e) 90%
HNO.sub.3, AcOH. Experimental Section:
[0695] The synthesis of SPI0027, SPI0028, SPI0029 and SPI0030 was
conducted in batches (3-10 g). Reagents mentioned in the
experimental section were purchased at the highest obtainable
purity from Lancaster, Sigma-Aldrich, Acros, or Bachem, except for
solvents, which were purchased from either Fisher Scientific or
Mallinkrodt.
1) SPI0027: Hydroxy-(2(S)-propyl-pentanoylamino)-acetic acid
(L-serine-valproic acid amide)
[0696] A) Preparation of SPI002701 (Protected Intermediate):
##STR85##
[0697] A mixture of 2-propylpentanoic acid (valproic acid 16.0 g,
0.11 mole) and thionyl chloride (30 mL) were stirred at room
temperature for 4 hours. After 4 hours, the solution was
concentrated under reduced pressure to prepare the acid chloride of
valproic acid (17.8 g, 98.8% yield). A portion of the remaining
colorless oil (11.0 g) was added drop-wise to an ice-cold mixture
of L-serine methyl ester, hydrochloride (10.60 g, 0.068 mole) and
triethylamine (30 mL) in anhydrous dichloromethane (100 mL). After
the addition, the mixture stirred for 2 hours under an argon
atmosphere, while cooling with an ice/water batch. The ice bath was
removed and the mixture stirred for 2 hours at room temperature.
The solvent and excess triethylamine were removed under reduced
pressure and a fresh aliquot of dichloromethane (100 mL) was added.
The mixture was then extracted with water (50 mL), 5% hydrochloric
acid (2.times.50 mL), saturated sodium bicarbonate solution
(2.times.50 mL), and DIUF water (50 mL). The remaining
dichloromethane solution was dried over sodium sulfate (10 g),
filtered, and concentrated under reduced pressure. The remaining
light yellow solid (17.22 g) was purified by column chromatography
on silica gel (350 g, 0.035-0.070 mm, 6 nm pore diameter), eluting
with hexanes/ethyl acetate (1:2). After concentration of the
product containing fractions under reduced pressure and drying
under high vacuum until the weight was constant, the experiment
produced the methyl ester protected L-serine-valproate amide
SPI002701 (9.45 g, 57% yield) as a white solid. ##STR86##
[0698] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=6.55 (1H, d,
J=7.2 Hz), 4.70 (1H, m), 4.0-3.89 (2H, m), 3.78 (3H, s), 3.16 (1H,
m), 2.17 (1H, m), 1.61 (2H, m), 1.46-1.23 (6H, m), 0.90 (6H, t,
J=7.2 Hz).
[0699] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=176.81, 171.08,
63.78, 54.67, 52.90, 47.61, 35.37, 21.01, 20.91, 14.38.
[0700] B) Deprotection of SPI002701: ##STR87##
[0701] The protected methyl ester protected L-serine-valproate
amide SPI002701 (9.40 g, 38.3 mmol) was dissolved in THF (100 mL)
and water (50 mL) containing lithium hydroxide (1.40 g, 58.4 mmol)
that was cooled in an ice-water batch. After stirring for 3 hours
while cooling in ice, 10% hydrochloric acid (50 mL) and the product
was extracted with dichloromethane (2.times.100 mL). The
dichloromethane fractions were combined, dried over sodium sulfate
(10 g), filtered and concentrated under reduced pressure. The crude
product (9.6 g, light yellow solid) was dissolved in diethyl ether
(40 mL) and stored overnight at -10.degree. C. After filtration,
the remaining white solid was dried at room temperature under high
vacuum until the product weight was constant (24 hours). The
experiment produced L-serine-valproic amide SPI0027 (5.62 g, 63%
yield) as a white solid. ##STR88##
[0702] .sup.1H NMR (300 MHz, DMSO): .delta.=12.3 (br s, 1H), 7.91
(d, 1H, J=7.5 Hz), 4.90 (br s, 1H), 4.28 (m, 1H), 3.63 (m, 2H),
2.82 (m, 1H), 1.42 (m, 2H), 1.21 (m, 6H), 0.82 (t, 6H, J=7 Hz).
[0703] .sup.13C NMR (75 MHz, DMSO): .delta.=174.92, 171.95, 61.51,
54.42, 44.85, 35.03, 34.88, 20.18, 20.03, 14.18, 14.13.
[0704] HPLC analysis:
[0705] 100% purity; r.t.=4.867 min.; 35% DIUF water (0.1% TFA)/65%
methanol; 1 mL/min; 39.8 C; Synergi Polar-RP 5 u column (serial #
234257), 4.6.times.250 mm; 20 ul injection; DAD1 A, Sig=210.4,
Ref=550,100.
[0706] CHN analysis:
[0707] calc. (C.sub.10H.sub.19NO4): C, 57.12; H, 9.15; N, 6.06.
found: C, 57.34; H, 9.22; N, 5.89.
[0708] Melting point: 65-67.degree. C.
2) SPI0028: 2-Propyl-pentanoic acid
4-(2(S)-amino-2-carboxy-ethyl)-phenyl ester, hydrochloride,
(L-Tyrosine-valproic acid ester, hydrochloride)
[0709] A) Preparation of SPI002801 (Protected Intermediate):
##STR89##
[0710] A mixture of 2-propylpentanoic acid (valproic acid, 2.66 g,
18.4 mmole), N-carbobenzyloxy-L-tyrosine benzyl ester (5.0 g, 12.3
mmole), EDC (3.54 g, 18.4 mmole), and DMAP (150 mg, 1.23 mmole) in
anhydrous dichloromethane (25 mL) was stirred under an argon
atmosphere at room temperature for 2 hours. After 2 hours, the
dichloromethane solution was washed with water (2.times.25 mL), 5%
hydrochloric acid (2.times.25 mL), 5% sodium bicarbonate
(2.times.25 mL), and DIUF water (25 mL). The dichloromethane was
dried over magnesium sulfate (5 g), filtered and concentrated under
reduced pressure. The remaining white solid (8.14 g) was purified
by column chromatography on silica gel (120 g), eluting with 3:1
heptane/ethyl acetate. The product containing fractions were
combined, washed with additional saturated sodium bicarbonate (100
mL, to remove valproic acid), and dried over sodium sulfate (10 g).
After concentration and drying to a constant weight, the experiment
gererated the protected intermediate SPI002801 (6.5 g, 99% yield)
as a white solid. ##STR90##
[0711] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.33 (10H, m),
6.98 (2H, d, J=8.4 Hz), 6.89 (2H, d, J=8.4 Hz), 5.26-5.09 (4H, m),
4.68 (1H, dd, J=13.5, 6.6 Hz), 3.09 (2H, m), 2.59 (1H, m),
1.80-1.36 (8H, m), 0.96 (6H, t, J=7.2 Hz).
[0712] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=174.86, 171.24,
155.64, 149.90, 135.06, 133.01, 130.34, 128.71, 128.59, 128.24,
128.18, 121.76, 67.50, 67.18, 54.99, 45.58, 45.22, 37.76, 34.92,
34.63, 20.97, 20.83, 14.33.
[0713] B) Deprotection of SPI002801: ##STR91##
[0714] The protected L-tyrosine-valproate ester SPI002801 (6.5 g)
was dissolved in ethyl acetate (100 mL) at room temperature and
added to a Parr bottle (500 mL) that contained 10% palladium on
carbon (2.25 g, 50% wet) under a nitrogen atmosphere. The nitrogen
atmosphere was replaced with hydrogen gas (32 psi). After 5 hours
of shaking, the ethyl acetate was acidified with 2N hydrogen
chloride in diethyl ether (20 mL) and the catalyst was removed by
filtration through a thin layer of celite 521 (20 g). The ethyl
acetate mixture was concentrated under reduced pressure at room
temperature. After drying for 2 hours under high vacuum at room
temperature, The remaining solid (5.4 g) was stirred in diethyl
ether (100 mL) overnight under an argon atmosphere. After
filtration and drying to a constant weight under high vacuum, the
experiment produced L-tyrosine-valproic acid ester SPI0028 (3.50 g,
72% yield) as a white solid. ##STR92##
[0715] .sup.1H NMR (300 MHz, DMSO): .delta.=8.63 (br s, 3H), 7.34
(d, 2H, J=8.4 Hz), 7.02 (d, 2H, J=8.4 Hz), 4.11 (br t, 1H), 3.18
(d, 2H, J=4.8 Hz), 2.57 (m, 1H), 1.66-1.45 (m, 4H), 1.45-1.29 (m,
4H), 0.91 (t, 6H, J=7.5 Hz).
[0716] .sup.13C NMR (75 MHz, DMSO): .delta.=174.02, 169.99, 149.36,
132.52, 130.62, 121.55, 53.16, 44.47, 34.95, 34.15, 20.19,
13.97.
[0717] HPLC analysis:
[0718] 99.75% purity; r.t.=7.433 min.; 60% DIUF water (0.1%
TFA)/40% acetonitrile; 1 mL/min; 39 C; Luna C18, 5 u column (serial
# 191070-3), 4.6.times.250 mm; 20 ul injection; DAD1 A, Sig=210,
Ref=550,100.
[0719] CHN analysis:
[0720] calc. (C17H26ClNO4): C, 59.38; H, 7.62; N, 4.07. found: C,
59.46; H, 7.58; N, 4.14.
[0721] Melting point: 192.0-194.0.degree. C.
3) SPI0029:
4(R)-Hydroxy-1-(2-propyl-pentanoyl)-pyrrolidine-2(S)-carboxylic
acid
(Valproic-L-hydroxyproline amide)
[0722] A) Preparation of SPI002901 (Protected Intermediate):
##STR93##
[0723] A mixture of 2-propylpentanoic acid (valproic acid 2.95 g,
0.02 mole) and thionyl chloride (8 mL) were stirred at room
temperature for 4 hours. After 4 hours, the solution was
concentrated under reduced pressure. The remaining colorless oil
was added drop-wise to an ice-cold mixture of L-hydroxyproline
benzyl ester, hydrochloride (Bachem, 6.2 g) and triethylamine (10
mL) in anhydrous dichloromethane (50 mL). After the addition, the
mixture stirred for 2 hours under an argon atmosphere, while
cooling with an ice/water batch. The ice bath was removed and the
mixture stirred for 4 hours at room temperature. The solvent and
excess triethylamine were removed under reduced pressure and a
fresh aliquot of dichloromethane (100 mL) was added. The mixture
was then extracted with water (50 mL), 5% hydrochloric acid
(2.times.50 mL), saturated sodium bicarbonate solution (2.times.50
mL), and DIUF water (50 mL). The remaining dichloromethane solution
was dried over sodium sulfate (10 g), filtered, and concentrated
under reduced pressure. The remaining crude product (6.2 g) was
purified by column chromatography on silica gel (110 g, 0.035-0.070
mm, 6 nm pore diameter), eluting with hexanes/ethyl acetate (1:1).
After concentration of the product containing fractions under
reduced pressure and drying under high vacuum until the weight was
constant, the experiment produced the protected
L-hydroxyproline-valproate amide SPI002901 (5.70 g, 80% yield) as a
colorless oil. ##STR94##
[0724] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.37-7.27 (5H,
m), 5.14 (2H, d, J=12.6 Hz), 4.63 (1H, t, J=7.8 Hz), 4.52 (1H, m),
3.76 (1H, dd, J=10.4, 4.2 Hz), 3.58 (1H, dd, J=10.4, 1.5 Hz), 2.47
(1H, m), 2.25 (1H, m), 2.04 (1H, m), 1.64-1.55 (2H, m), 1.43-1.19
(6H, m), 0.8 (6H, m).
[0725] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=175.96, 172.21,
135.67, 128.56, 128.23, 70.15, 67.00, 58.02, 55.33, 43.56, 37.76,
35.44, 35.08, 21.02, 20.71, 14.51.
[0726] The protected L-hydroxyproline-valproate ester SPI002901
(9.3 g, 26.7 mmole) was dissolved in ethyl acetate (25 mL) at room
temperature and added to a Parr bottle (500 mL) that contained 10%
palladium on carbon (1.53 g, 50% wet) under a nitrogen atmosphere.
The nitrogen atmosphere was replaced with hydrogen gas (35 psi).
After 20 hours the catalyst was removed by filtration through a
thin layer of activated carbon, washing with ethanol (25 mL). The
ethanol and ethyl acetate were concentrated under reduced pressure
at room temperature. After drying under high vacuum, the remaining
solid (6.54 g) was crystallized from t-butyl methyl ether (100 mL,
48 hours at -10.degree. C.). After filtration and drying to a
constant weight, the experiment produced L-hydroxyproline-valproic
amide SPI0029 (5.17 g, 83% yield) as a white solid.
[0727] .sup.1H NMR (300 MHz, DMSO): .delta.=12.6 (br s, 1H), 5.39
(br s, 1H), 4.69-4.48 (m, 2H), 3.90-3.58 (m, 4H), 2.78 (m, 1H),
2.51-2.32 (m, 2H), 2.22-2.12 (m, 1H), 1.80-1.42 (m, 8H), 1.11 (m,
6H).
[0728] .sup.13C NMR (75 MHz, DMSO): .delta.=173.80, 173.43, 68.78,
75.42, 55.04, 42.10, 37.29, 35.13, 34.74, 20.17, 19.74, 14.28,
14.24.
[0729] HPLC analysis:
[0730] 99.89% purity; r.t.=7.20 min.; 70% DIUF water (0.1% TFA)/30%
ACN; 1 mL/min; 39.8 C; Synergi Polar-RP 5 u column (serial #
161309), 4.6.times.250 mm; 20 ul injection; DAD1 A, Sig=210.4,
Ref=550,100.
[0731] Specific Rotation: -56.8 (5 mg/mL in ethanol at 25 C)
[0732] CHN analysis:
[0733] calc. (C13H23NO4): C, 60.68; H, 9.01; N, 5.44. found: C,
60.71; H, 9.03; N, 5.45.
[0734] Melting point: 113-114.degree. C. 4) SPI0030:
3-Nitroxy-2(S)-(2-propyl-pentanoylamino)-propionic acid
(L-serine(O-nitroxy ester)-valproic acid amide) ##STR95##
[0735] The L-serine valproic amide (SPI0029, 8.4 g, 0.036 mole) was
dissolved in glacial acetic acid (60 mL). The flask was cooled in
an ice/water bath under an argon atmosphere and cold 90% nitric
acid (6.0 mL) was added. After 4 hours at 5.degree. C., the
solution was poured into ice (400 g) and extracted with
dichloromethane (2.times.150 mL). The dichloromethane fractions
were combined, dried over sodium sulfate (15 g), filtered and
concentrated. Toluene (150 mL) was added to the remaining oil and
the solution was concentrated a second time to remove acetic acid.
After drying under high vacuum to a constant weight, the remaining
oil (12.4 g) was dissolved in toluene (50 mL) and stored at
-10.degree. C. overnight. After 72 hours at -10.degree. C., the
solids were filtered and dried to a constant weight. The experiment
produced the nitroxy ester of the L-serine-valproic amide SPI0030
(4.37 g, 43% yield) as a white solid. ##STR96##
[0736] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=8.88 (br s, 1H),
6.60 (d, 1H, J=7.2 Hz), 5.00 (m, 1H), 4.87 (m, 2H), 2.23 (m, 1H),
1.56 (m, 2H), 1.43 (m, 2H), 1.29 (m, 6H), 0.89 (t, 6H, J=7.2
Hz).
[0737] .sup.13C NMR (75 MHz, CDCl): .delta.=177.83, 170.88, 71.42,
50.60, 47.47, 35.15, 20.88, 14.28.
[0738] HPLC analysis:
[0739] 99.25% purity; r.t.=5.400 min.; 65% methanol/35% phosphate
buffer (pH=3); 1 mL/min; 36.0 C; Synergi Polar-RP 5 u column
(serial # 161309-2), 4.6.times.250 mm; 20 ul injection;
Sig=210.4.
[0740] Specific Rotation: -19.7 (10.1 mg/mL in ethanol at 25 C)
[0741] CHN analysis:
[0742] calc. (C11H20N2O6): C, 47.82; H, 7.30; N, 10.14. found: C,
48.12; H, 7.19; N, 9.84.
[0743] Melting point: 49.0-51.0.degree. C.
[0744] Solubility of the above esters were determined in water at
room temperature by dissolving excess of each of the drug and
allowing them to settle for a few hours. The resulting solutions
were centrifuged at 1500 rpm for 3 min and the supernatant liquid
was analyzed. It was shown that these esters possess solubility in
water in excess of 50 mg/mL.
[0745] There are a number of screening tests to determine the
utility of the derivatives created according to the disclosed
methods. These include both in vitro and in vivo screening
methods.
[0746] The in vitro methods include acid/base hydrolysis of the
derivatives, hydrolysis in pig pancreas, hydrolysis in rat
intestinal fluid, hydrolysis in human gastric fluid, hydrolysis in
human intestinal fluid, and hydrolysis in human blood plasma. These
assays are described in Simmons, D M, Chandran, V R and Portmann, G
A, Danazol, Amino Acid Derivatives: In Vitro and In Situ
Biopharmaceutical Evaluation, Drug Development and Industrial
Pharmacy, Vol. 21, Issue 6, Page 687, 1995, the contents of all of
which are incorporated by reference.
[0747] The amino acid derivatives of Valproic acid of the present
invention are effective in treating diseases or conditions in which
Valproic acid normally is used. Without wishing to be bound, it is
believed that the amino acid derivatives disclosed herein are
transformed within the body to release the active compound.
Alternatively, the amino acid derivative may not be transformed to
release the active component in the body. This latter form enhances
the therapeutic benefits of the Valproic acid by reducing or
eliminating biopharmaceutical and pharmacokinetic barriers
associated with each of them. However it should be noted that these
derivatives themselves will have sufficient activity without
releasing any active drug in the mammals.
[0748] Thus, the amino acid derivative of the present invention
enhances the therapeutic benefits by removing biopharmaceutical and
pharmacokenetic barriers of existing drugs. Furthermore, the
derivatives are easily synthesized in high yields using reagents
which are readily and commercially available.
Synthesis of Clopidogrel Derivatives:
Overview:
[0749] The procedure for the synthesis of the L-serine and
L-threonine esters of clopidogrel is outlined in Synthetic Sequence
section. The complete procedure and analytical data are given in
the Experimental Section. In general, the hydrosulfate salt of
clopidogrel (Xiangding Chemical International Company) was treated
with sodium bicarbonate to generate the free amine. The methyl
ester was then removed by displacement with lithium iodide in
pyridine. The unwanted isomer generated in the reaction was
partially removed by crystallization from water. The acid
intermediate was then coupled with the tert-butyl ester of
boc-L-serine or boc-L-threonine using EDC. The protected
intermediate esters were purified twice by column chromatography
and treated with dilute hydrochloric acid in acetic acid at low
temperature to remove the protective groups. Washing the final
salts with ethyl acetate purified the amino acid ester salts of
clopidogrel. The final salts were dried under high vacuum and
shipped to Signature Pharmaceuticals Inc., after analysis by NMR,
HPLC, CHN, specific rotation, and melting point. Synthetic
Sequence: ##STR97## Synthesis of the L-serine and L-threonine
Esters of Clopidogrel: a) NaHCO.sub.3; b) LiI, pyridine; c)
boc-THR-(OtBu), EDC, DMAP; d) boc-SER-(OtBu), EDC, DMAP; e) 1 M
HCl, AcOH. Experimental Section:
[0750] The syntheses of SPIB00301 and SPIB00302 were conducted in
batches. Reagents mentioned in the experimental section were
purchased at the highest obtainable purity from Lancaster,
Sigma-Aldrich, Acros, Bachem, or Xiangding Chemical International,
except for solvents, which were purchased from either Fisher
Scientific or Mallinkrodt. 1) Preparation of Clopidogrel Acid:
##STR98##
[0751] To a solution of clopidogrel hydrosulfate (97.7 g, 233 mmol)
in DIUF water (1 L) was added sodium bicarbonate (39.1 g, 466 mmol)
in small portions. After mixing, t-butyl methyl ether (1 L) was
added and the solution stirred for 30 minutes. The layers were
separated and the aqueous layer was extracted a second time with
t-butyl methyl ether (300 mL). The organic layers were combined,
washed with brine (500 mL), and dried over sodium sulfate. After
filtration, the t-butyl methyl ether was removed under reduced
pressure. The remaining clopidogrel (yellow oil, 77.82 g, 104%
yield) was dried under high vacuum at room temperature for 18 hours
until most of the t-butyl methyl ether was removed. The clopidogrel
(77.8 g, 242 mmol) was dissolved in pyridine (200 mL) and added to
a refluxing solution of anhydrous lithium iodide (120 g, 897 mmol)
in pyridine (800 mL). After 8 hours at reflux, the flask was cooled
to room temperature and the pyridine was removed under reduced
pressure (at 40.degree. C.). The remaining wet solid (470 g) was
dissolved in DIUF water (300 mL) and acidified to pH=5.5 with
acetic acid (50 mL). The product was then extracted with
dichloromethane (3.times.300 mL). The dichloromethane fractions
were combined, dried over sodium sulfate, filtered, concentrated
under reduced pressure, and dried under high vacuum. The remaining
yellow mixture (161.3 g) was mixed with anhydrous THF (500 mL) for
60 minutes. After 60 minutes, the salts were removed by filtration.
The filtrate was concentrated under reduced pressure to leave a
brown oil (128.3 g), which was dissolved in DIUF water (3.times.200
mL) and freeze-dried under high vacuum to remove traces of solvent.
The remaining yellow solid (108.5 g) was again added to water (600
mL) and sonicated. The solids were removed by filtration and the
filtrate was freeze-dried under high vacuum. The remaining solid
(68.7 g) was partitioned between water (400 mL) and t-butyl methyl
ether (400 mL), stirred for 24 hours, stored over the weekend at
5.degree. C., and filtered. The layers were separated and the
aqueous layer was extracted a second time with t-butyl methyl ether
(300 mL). The purified product was then extracted from the aqueous
layer with dichloromethane (2.times.200 mL). The dichloromethane
fractions were combined, dried over sodium sulfate, filtered,
concentrated and freeze-dried (from 100 mL DIUF water). The
procedure generated the acid of clopidogrel (28.5 g) as a yellow
solid.
[0752] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 8.02 (1H, s br),
7.88 (1H, d, J=7.5 Hz), 7.37 (1H, d, J=8.1 Hz), 7.22 (1H, t, J=7.5
Hz), 7.13 (2H, m), 6.65 (1H, d, J=4.8 Hz), 5.14 (1H, s), 4.14 (2H,
m), 3.37 (1H, m), 3.28 (1H, m), 3.00 (2H,m).
[0753] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 169.75, 135.08,
131.71, 131.49, 130.07, 129.98, 129.82, 129.08, 127.44, 125.06,
124.14, 67.23, 50.22, 48.30, 22.62.
[0754] Specific rotation: +43.5 deg (23.degree. C., 0.0216 g/2 mL
methanol, 589 nm)
2) Preparation of the L-threonine Ester of Clopidogrel:
[0755] Coupling Procedure: ##STR99##
[0756] A mixture of the acid of clopidogrel (14.1 g, 45.8 mmol),
boc-L-threonine tert-butyl ester (11.4 g, 41.6 mmol, prepared by
the literature method),
1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI,
7.38 g, 38.5 mmol), and DMAP (0.783 g, 6.4 mmol) in anhydrous
dichloromethane (300 mL) was stirred at room temperature under an
argon atmosphere for 22 hours. Additional dichloromethane (1.2 L)
was added and the dichloromethane solution was washed with water
(1200 mL) and 5% sodium bicarbonate solution (1200 mL). After
drying the dichloromethane solution over sodium sulfate,
filtration, and concentration under reduced pressure, the residue
(24 g) was purified by column chromatography on silica gel (500 g),
eluting with heptane-ethyl acetate (8:1). The product containing
fractions were combined and concentrated to provide the protected
L-threonine ester of clopidogrel SPIB0030101 (13.5 g, 52% yield),
as clear liquid.
[0757] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.69 (1H, m),
7.42 (1H, dd, J=7.2, 2.1 Hz), 7.29 (2H, m), 7.06 (1H, d, J=5.1 Hz),
6.65 (1H, d, J=5.1 Hz), 5.47 (1H, m), 5.16 (1H, m), 4.84 (1H, s),
4.30 (1H, d, J=3.6 Hz), 3.65 (2H, m), 2.85 (4H, m), 1.46 (18H, m),
1.15 (3H, d, J=6 Hz).
[0758] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 169.46, 168.91,
155.89, 134.63, 133.68, 133.23, 129.96, 129.85, 129.52, 127.27,
125.22, 122.80, 82.61, 80.14, 71.62, 68.16, 57.65, 50.72, 48.51,
28.50, 27.89, 25.65, 16.84. Deprotection and Purification:
##STR100##
[0759] A solution of hydrogen chloride (1.0 M in acetic acid, 210.6
mL) was added drop-wise to a solution of the protected L-threonine
ester of clopidogrel SPIB0030101 (11.9 g, 21.06 mmol) in anhydrous
dichloromethane (115 mL) that was cooled in an ice batch, under an
argon atmosphere. The mixture was stored for 6.5 days at 5.degree.
C. The mixture was concentrated under reduced pressure and dried
under high vacuum to generate a light, yellow solid (13.22 g).
Ethyl acetate (150 mL) was added to the solid and the mixture was
sonicated for 5 minutes. The ethyl acetate was removed by
filtration and the remaining solid was added to an additional
volume of ethyl acetate (150 mL) and the mixture was sonicated a
second time for 5 minutes. After filtration the sonication process
was repeated a third time. The white, solid product (10.5 g) was
filtered and dissolved in dichloromethane (320 mL). Ethyl acetate
(150 mL) was added and the solution was stored at -20.degree. C.
overnight. The solids were filtered and the crystallization process
was repeated with methanol (1.5 mL)/dichloromethane (180 mL) and
ethyl acetate. The solids were filtered and freeze-dried under high
vacuum. The procedure generated the clopidogrel-L-threonine ester,
dihydrochloride SPIB00301 (6.1 g, 60% yield, 99.51% purity by HPLC)
in monohydrate form (as white solid).
[0760] .sup.1H NMR (300 MHz, DMSO): .delta. 7.78-7.58 (m, 4H), 7.42
(m, 1H), 6.80 (m, 1H), 5.95 (m, 1H), 5.85 (m, 1H), 4.43 (m, 1H),
4.31-4.17 (m, 3H), 3.84 (m, 2H), 3.33 (m, 2H), 1.33 (d, 3H, J=6
Hz).
[0761] .sup.13C NMR (75 MHz, DMSO): .delta. 170.56, 167.11, 136.33,
134.39, 132.46, 129.93, 127.46, 126.66, 126.51, 126.01, 73.09,
66.88, 62.91, 57.94, 51.81, 22.93, 16.75.
[0762] HPLC analysis:
[0763] 99.51% purity, r.t.=10.042 min, sample dissolved in DIUF
water/ACN, 75% DIUF water (0.1% TFA)/25% ACN, Hydro-RP (#162383-7),
4 u, 250.times.4.6 mm, 1 mL/min., 40.degree. C., 20 uL inj. vol.,
SPD-10 Avp, chl-210 nm.
[0764] Melting point: 110-112.degree. C. (Thomas Hoover CMPA,
uncorrected)
[0765] Specific rotation: +22.5 deg (25.degree. C., 27.7 mg/5 mL
ethanol 589 nm)
[0766] CHN analysis:
[0767] calc.: C, 45.66; H, 5.04; N, 5.60; Cl, 21.28.
(C.sub.19H.sub.25Cl.sub.3N.sub.2O.sub.5S); found: C, 44.89; H,
4.96; N, 5.61; Cl, 21.32.
3) Preparation of the L-serine Ester of Clopidogrel:
[0768] Coupling Procedure: ##STR101##
[0769] A mixture of the acid of clopidogrel (10.24 g, 33.2 mmol),
boc-L-serine tert-butyl ester (7.80 g, 29.8 mmol),
1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI,
8.94 g, 46.6 mmol), and DMAP (0.25 g) in anhydrous dichloromethane
(200 mL) was stirred at room temperature under an argon atmosphere
for 4 hours. The dichloromethane solution was washed with water
(2.times.200 mL) and saturated sodium bicarbonate (250 mL). After
drying over sodium sulfate, filtration, and concentration under
reduced pressure, the remaining yellow oil (18.62 g) was purified
twice by column chromatography on silica gel (300 g), on it eluting
with heptane-ethyl acetate (9:1). The product containing fractions
were combined in two fractions and concentrated. The procedure
generated the protected L-serine ester of clopidogrel SPIB0030201
(6.32 g, 34% yield).
[0770] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.67 (1H, m),
7.42 (1H, m), 7.29 (2H, m), 7.06 (1H, d, J=5.1 Hz), 6.66 (1H, d,
J=5.1 Hz), 4.95 (1H, d, J=7.8 Hz), 4.89 (1H, s), 4.44 (3H, d), 3.72
(1H, d, J=14.4 Hz), 3.60 (1H, d, J=14.4 Hz), 2.87 (4H, m), 1.42
(18H, m).
[0771] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 170.16, 168.19,
154.79, 134.48, 133.61, 133.11, 129.81, 129.75, 129.42, 127.13,
125.10, 122.64, 82.72, 79.94, 71.62, 67.77, 64.71, 53.46, 50.66,
48.39, 28.36, 27.97, 25.55. Deprotection and Purification:
##STR102##
[0772] A solution of hydrogen chloride (1.0 M in acetic acid, 100
mL) was added drop-wise to a solution of the protected L-serine
ester of clopidogrel SPIB0030201 (6.30 g, 11.43 mmol) in anhydrous
dichloromethane (100 mL) that was cooled in an ice batch, under an
argon atmosphere. The mixture was stored for 4 days at 5.degree. C.
The mixture was concentrated under reduced pressure and dried under
high vacuum to generate a colorless gel. Ethyl acetate (250 mL) was
added to the gel and the mixture was sonicated for 5 minutes. The
white, solid product was filtered and dried under high vacuum. The
white, solid product (6.4 g) was filtered and dissolved in
dichloromethane (250 mL). Ethyl acetate (150 mL) was added and the
solution was stored at -20.degree. C. overnight. After filtration
and freeze-drying, the procedure generated the clopidogrel-L-serine
ester, dihydrochloride SPIB00302 (4.8 g, 97% yield, 96.64% purity
by HPLC) in monohydrate form (as off-white solid).
[0773] .sup.1H NMR (300 MHz, DMSO): .delta. 9.18 (br s, 3H), 8.16
(d, 1H, J=6.6 Hz), 7.66-7.40 (m, 4H), 6.87 (m, 1H), 5.76 (br s,
1H), 4.75 (m, 2H), 4.49 (m, 1H), 4.40-4.0 (m, 1H). 3.40-3.80 (m,
1H), 3.17 (m, 2H).
[0774] .sup.13C NMR (75 MHz, DMSO): .delta. 167.50, 164.29, 134.23,
132.29, 131.35, 130.93, 130.41, 128.26, 127.81, 126.98, 125.33,
124.73, 66.12, 63.95, 50.82, 50.15, 49.12, 22.13.
[0775] HPLC analysis:
[0776] 96.64% purity, r.t.=10.56 min, sample dissolved in DIUF
water/ACN, 83% DIUF water (0.1% TFA), 17% ACN, Gemini-C18
(#262049-2), 5 u, 250.times.4.6 mm, 1 mL/min., 40.degree. C., 20 uL
inj. vol., SPD-10 Avp, chl-210 nm.
[0777] Specific rotation: +42.0 deg (25.degree. C., 34.0 mg/5 mL
ethanol 589 nm)
[0778] Melting point: 100-102.degree. C. (Thomas Hoover CMPA,
uncorrected)
[0779] CHN analysis:
[0780] calc.: C, 44.50; H, 4.77; N, 5.77; Cl, 21.89.
(C.sub.18H.sub.23Cl.sub.3N.sub.2O.sub.5S); found: C, 44.63; H,
4.69; N, 5.76; Cl, 22.20.
Synthesis of Danazol Proline Ester
Overview:
[0781] The procedure for the synthesis of the L-proline esters of
Danazol is outlined in Synthetic Sequence section. The complete
procedure and analytical data is given in the Experimental Section.
In general, Danazol (4-7 g, in batches) was coupled with
N-tert-butoxycarbonyl-L-proline(1.2-1.6 equivalents) with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,
1.2-1.4 equivalents) in the presence of catalytic amount of
4-(N,N-dimethylamino)-pyridine (DMAP). Once the reaction was
complete, the excess EDC and N-tert-butoxycarbonyl-L-proline were
removed by extraction. The crude protected ester of Danazol was
purified by column chromatography on silica gel. Unreacted Danazol
was collected and reprocessed. The yield for the coupling step was
80-96%. The protective group was removed with 2M hydrogen chloride
solution in diethyl ether at low temperature. The hydrochloride
salt of the Danazol-L-proline ester was purified as the free base
by column chromatography. Once pure, the free base of
Danazol-L-proline ester was converted to the hydrochloride salt
with diluted hydrogen chloride solution in diethyl ether, at low
temperature. Synthetic Sequence: ##STR103## Synthesis of the
L-proline Ester of Danazol: a) EDC, DMAP, CH.sub.2Cl.sub.2; b) HCl,
Diethyl Ether; c) NaHCO3, CH.sub.2Cl.sub.2; d) HCl,
CH.sub.2Cl.sub.2, Diethyl Ether. Experimental Section:
[0782] The synthesis of SPIC004 was conducted in several small
batches. A representative batch for each step is described below.
Reagents mentioned in the experimental section were purchased at
the highest obtainable purity from Spectrum, Fluka, Acros and
Sigma-Aldrich. 1) Synthesis of SPC00401:
Danazol-N-tert-butoxycarbonyl-L-proline ester Danazol (7.0 g, 20.74
mmole), N-tert-butoxycarbonyl-L-proline (4.55 g, 21.14 mmole),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,
4.0 g, 20.86 mmole) and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.27
g, 2.2 mmole) were dissolved in anhydrous dichloromethane (28 ml)
while stirring and cooling in an ice-water bath. The ice bath was
removed and the mixture was allowed to stir at room temperature for
24 hours. After stirring under an argon atmosphere at room
temperature for 24 hours, additional
N-tert-butoxycarbonyl-L-proline (0.9 g, 4.18 mmole), EDC (0.8 g,
4.17 mmole) and DMAP (0.06 g, 0.5 mmole) were added to the reaction
mixture. After 6 days of stirring under an argon atmosphere at room
temperature, additional reagents were added:
N-tert-butoxycarbonyl-L-proline (0.9 g, 4.18 mmole), EDC (0.8 g,
4.17 mmole) and DMAP (0.06 g, 0.5 mmole). After 8 days, the
reaction mixture was diluted with dichloromethane (40 mL), and
extracted with saturated sodium bicarbonate solution (2.times.100
mL) and DIUF water (3.times.100 mL). After drying over anhydrous
sodium sulfate, the solution was filtered and concentrated under
reduced pressure. The light yellow solid foam (12.6 g) that
remained was purified by column chromatography on silica gel (450
g, 0.035-0.070 mm, 6 nm pore diameter), eluting with toluene:
acetone (9:1). The product containing fractions were combined,
concentrated under reduced pressure, and dried under high vacuum
until a constant weight was obtained. The experiment produced the
protected Danazol-L-proline ester (SPIC00401) as a white, solid
foam (7.8 g, 70.3% yield). A second fraction containing a mixture
of Danazol and the protected Danazol-L-proline ester (3.7 g) was
also recovered. The recovered mixture was reprocessed (as above) to
produce an additional amount of SPIC00401 (2.88 g, 26.0%).
##STR104##
Pyrrolidine-1,(2S)-dicarboxylic acid 1-tert-butyl ester
2-(1-ethynyl-10a,12a-dimethyl-2,3,3a,3b,4,5,10,10a,10b,11,12,12a-dodecahy-
dro-1H-7-oxa-8-aza-dicyclopenta[a,h]phenanthren-1-yl)ester
[0783] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=8.00 (1H, s),
6.17 (1H, s), 4.32-4.20 (1H, m), 3.58-3.35 (2H, m), 2.84-1.0 (37H,
m). (Mixture of conformational isomers)
[0784] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=171.52, 171.21,
164.82, 154.27, 154.09, 153.77, 148.68, 108.94, 107.68, 85.06,
84.85, 82.94, 80.07, 79.71, 75.57, 75.15, 59.40, 58.92, 53.87,
48.47, 48.36, 47.69, 46.66, 46.45, 41.18, 37.51, 36.84, 33.54,
33.08, 32.38, 31.07, 30.89, 29.77, 28.69, 28.57, 24.39, 23.95,
23.72, 21.72, 18.97, 13.77. (Mixture of conformational isomers)
2) Synthesis of SPIC004: Danazol-L-proline Ester, Hydrochloride
[0785] To a stirred solution of protected Danazol-L-proline ester
SPIC00401 (7.8 g, 14.58 mmole) in anhydrous diethyl ether (32 mL)
cooled in an ice-water bath, a solution of hydrogen chloride in
diethyl ether (73 mL, 2M, 146 mmole) was added drop-wise under an
argon atmosphere. After 4 days stirring at room temperature, the
mixture was concentrated under reduced pressure and dried under
high vacuum. To the remaining yellow solid (6.68 g) was added
anhydrous diethyl ether (70 mL). The mixture was stirred at room
temperature for 20 hours under an argon atmosphere. Crude
Danazol-L-proline ester, hydrochloride SPIC004 (4.6 g, 67.0% yield,
HPLC purity 94%) was isolated as a light, yellow solid.
3) Conversion and Purification of SPIC004b: Danazol-L-proline
Ester
[0786] To a cold solution of Danazol-L-proline ester, hydrochloride
SPIC004 (10.45 g, 22.18 mmole) in DIUF water (100 mL) was added
dichloromethane (100 mL). Solid sodium bicarbonate (3.72 g, 44.36
mmole) was added in portions. The layers were separated and the
aqueous layer was extracted with dichloromethane (2.times.50 mL).
The organic layer was dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure. The remaining
oil (12.1 g) was purified by column chromatography on silica gel,
eluting with 2-propanol. The product containing fractions were
combined and concentrated under reduced pressure. The remaining
yellow solid foam (8.44 g) was dissolved at room temperature in
anhydrous diethyl ether (85 mL) and kept at -10.degree. C. for 24
hours. After filtration of the solid product and drying under high
vacuum, Danazol-L-proline ester SPIC004b (5.89 g, 61.0% yield) as a
light yellow solid (powder) was isolated. ##STR105##
Pyrrolidine-(2S)-carboxylic acid 1-ethynyl-10a,
12a-dimethyl-2,3,3a,3b,4,5,10,10a,10b,11,12,12a-dodecahydro-1H-7-oxa-8-az-
a-dicyclopenta[a,h]phenanthren-1-yl ester
[0787] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=8.00 (1H, s),
6.18 (1H, s), 3.73 (1H, m), 3.10 (1H, m), 2.89 (1H, m), 2.76-2.70
(2H, m), 2.62-2.35 (3H, m), 2.25-1.07 (18H, m), 1.02 (3H, s), 0.91
(3H, s).
[0788] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=173.93, 164.83,
154.16, 148.64, 108.91, 107.66, 85.09, 82.97, 75.30, 59.94, 53.85,
48.49, 47.73, 47.17, 41.17, 37.53, 36.85, 33.54, 33.26, 32.37,
30.88, 30.31, 25.63, 23.91, 21.38, 18.98, 13.67.
4) Regeneration of SPIC004: Danazol-L-proline Ester,
Hydrochloride
[0789] Hydrogen chloride solution (26.8 mL, 1M, 26.8 mmole) was
added drop-wise, under an argon atmosphere, to a stirred solution
of Danazol-L-proline ester SPIC004b (5.8 g, 13.35 mmole) in
anhydrous dichloromethane (30 mL), cooled at -15.degree. C. After
stirring for 4 hours in an ice-water bath, the suspension was
filtered, the solid dried under high vacuum at room temperature
until the weight was constant. The procedure generated the purified
Danazol-L-proline ester, hydrochloride SPIC004 as a very light
yellow solid (5.62 g, 89.3%, HPLC purity 98.2%). ##STR106##
Pyrrolidine-(2S)-carboxylic acid
1-ethynyl-10a,12a-dimethyl-2,3,3a,3b,4,5,10,10a,10b,11,12,12a-dodecahydro-
-1H-7-oxa-8-aza-dicyclopenta[a,h]phenanthren-1-yl ester,
hydrochloride
[0790] .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta.=10.40 (1H, br
s), 9.17 (1H, br s), 8.34 (1H, s), 6.28 (1H, s), 4.36 (1H, dd,
J=7.2, 6.9 Hz), 3.76 (1H, s), 3.22 (2H, m), 2.73 (1H, d, J=15.9
Hz), 2.60-2.32 (7H, m), 2.0-1.35 (12H, m), 1.13 (1H, m), 0.97 (3H,
s), 0.93 (3H, s).
[0791] .sup.13C NMR (75 MHz, DMSO-d.sub.6): .delta.=166.79, 163.73,
154.27, 148.86, 108.06, 107.49, 85.95, 82.09, 78.68, 58.55, 53.15,
47.97, 47.26, 45.11, 40.44, 36.41, 35.86, 32.69, 32.52, 31.35,
30.33, 27.73, 23.23, 22.89, 20.63, 18.49, 13.26.
[0792] HPLC analysis: 98.27% purity, r.t.=14.48 min, sample
dissolved in DIUF water/ACN, 50% DIUF water (0.1% TFA)/50% ACN,
Gemini C18 (#262049-2), 5 u, 250.times.4.6 mm, 1 mL/min.,
40.degree. C., 20 uL inj. vol., SPD-10 Avp, chl-210 nm.
[0793] CHN analysis: calculated: C, 68.85; H, 7.49; N, 5.95; Cl,
7.53. found: C, 68.66; H, 7.49; N, 5.89; Cl, 7.76.
[0794] Specific rotation: -6.67 deg (20.degree. C., 19.5 mg/l mL
ethanol, 589 nm)
[0795] Melting point: 195.0-198.0.degree. C. (dec.)
Preparation of Benazepril Esters
Overview:
[0796] The procedure for the synthesis of the L-serine,
L-threonine, and L-hydroxyproline esters of benazepril is outlined
in Synthetic Sequence section. The complete procedure and
analytical data are given in the Experimental Section. In general,
benazepril (Xiangding Chemical International Company) was butylated
to protect the free acid group. The ethyl ester was then hydrolyzed
to the acid with sodium hydroxide. The amine present in the
molecule was protected with a tert-butyloxycarbonyl group to
prevent dimerization during the subsequent coupling reaction. The
protected intermediate was then coupled with the tert-butyl ester
of boc-L-serine, boc-L-threonine, or boc-L-hydroxyproline using
EDC. The protected intermediate esters were treated with dilute
hydrochloric acid in acetic acid at low temperature to remove all
of the protecting groups. The final amino acid ester salts of
benazepril were purified by washing with ethyl acetate and
dichloromethane, dried under high vacuum, and analyzed by NMR,
HPLC, CHN, specific rotation, and melting point. Synthetic
Sequence: ##STR107## ##STR108## Synthesis of the L-serine,
L-threonine, and L-hydroxyproline Esters of Benazepril: a) THF; b)
NaOH, MeOH, H.sub.2O; c) (Boc).sub.2O, Et.sub.3N, CH.sub.2Cl.sub.2;
d) Boc-Ser-OtBu, EDC, DMAP, CH.sub.2Cl.sub.2; e) Boc-Thr-OtBu, EDC,
DMAP, CH.sub.2Cl.sub.2; f) Boc-Hyp-OtBu, EDC, DMAP,
CH.sub.2Cl.sub.2; g) HCl, AcOH, CH.sub.2Cl.sub.2. Experimental
Section:
[0797] The synthesis of SPIB00501, SPIB00502 and SPIB00503 was
conducted in batches. Reagents mentioned in the experimental
section were purchased at the highest obtainable purity from
Lancaster, Sigma-Aldrich, Acros, Bachem, or Xiangding Chemical
International, except for solvents, which were purchased from
either Fisher Scientific or Mallinkrodt. 1) Butylation of
Benazepril: ##STR109##
[0798] A solution of 2-tert-butyl-1,3-dicyclohexyl isourea 1 (569
g, 2.03 mol, prepared by the literature procedure) in THF (500 mL)
was added drop-wise to a mixture of benazepril hydrochloride (117
g, 0.254 mol) in anhydrous THF (500 mL) and cooled in an ice-water
bath. After 2 hours, the ice bath was removed and the mixture was
allowed to stir for 4 days at room temperature under an argon
atmosphere. The reaction mixture was filtered and the precipitate
(DCU) was washed with methyl t-butyl ether (3.times.500 mL). The
filtrates were combined and washed with 5% sodium bicarbonate
solution (1 L) and brine (1 L). The ether-THF solution was dried
over sodium sulfate, filtered, and concentrated under reduced
pressure. The remaining mixture (202 g) was purified by column
chromatography on silica gel (1 Kg), eluting with heptane-ethyl
acetate (4:1). The product containing fractions were combined and
concentrated under reduced pressure and dried at high vacuum till a
constant weight was achieved and a butylated benazepril 2 (88.17 g,
72% yield) was isolated as a yellow oil.
[0799] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.18 (9H, m),
4.61 (1H, d, J=16.8 Hz), 4.31 (1H, d, J=16.8 Hz), 4.04 (2H, m),
3.25 (3H, m), 2.69 (2H, m), 2.57 (1H, m), 2.39 (1H, m), 2.19 (1H,
s, br), 1.99 (3H, m), 1.43 (9H, s), 1.11 (3H, t, J=7.2 Hz).
[0800] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 173.89, 173.58,
167.59, 141.21, 140.83, 135.91, 129.27, 128.21, 128.16, 127.50,
126.53, 125.71, 122.04, 81.88, 60.48, 60.18, 56.90, 51.16, 37.81,
35.13, 32.10, 28.52, 28.01, 14.14. 2) Saponification of the
Butylated Benazepril: ##STR110##
[0801] A sodium hydroxide solution (8.08 g, 0.202 mol) in DIUF
water (540 mL) was added in a drop-wise fashion to the butylated
benazepril 2 (88.17 g, 0.184 mol) dissolved in methanol (1.6 L),
while cooling the methanol solution in ice bath. The solution was
allowed to stir for 18 hours under an argon atmosphere while
cooling in an ice bath. The reaction mixture was allowed to warm to
room temperature and stir at room temperature for an additional 24
hours. The reaction mixture was concentrated under reduced pressure
and dried under high vacuum to produce a mixture of solid and
liquid (130 g). The residue was cooled in an ice bath and a
solution of acetic acid in water (1:10 v/v acetic acid/water) was
added drop-wise to adjust the solution to pH=7. A white precipitate
formed which was filtered overnight in two crops (141.3 g and 7.05
g). The combined precipitates fractions were stirred with
dichloromethane (500 mL) for one hour. After filtration and drying
under high vacuum, 3 (48.6 g, 59% yield) was isolated as a white
solid.
[0802] .sup.1H NMR (300 MHz, CD.sub.3OD): .delta. 7.34-7.16 (9H,
m), 4.65 (1H, d, J=17.4 Hz), 4.44 (1H, d, J=17.4 Hz), 3.87 (1H, dd,
J=11.4, 7.8 Hz), 3.45 (1H, t, J=5.7 Hz), 3.37 (1H, dd, J=12.9, 5.4
Hz), 2.74 (3H, m), 2.60 (1H, m), 2.34 (1H, m), 2.11 (2H, m), 1.43
(9H, s).
[0803] .sup.13C NMR (75 MHz, CD.sub.3OD): .delta. 172.67, 168.85,
168.71, 142.04, 140.99, 135.75, 130.65, 129.51, 129.38, 129.24,
128.69, 127.05, 123.88, 83.38, 63.51, 59.07, 52.20, 34.95, 34.29,
32.47, 28.40, 28.27. 3) Amine Protection: ##STR111##
[0804] A solution of di-tert-butyl dicarbonate (38.2 g, 175 mmol)
in anhydrous dichloromethane (200 mL) was added in a drop-wise
fashion to a solution of acid 3 (72.0 g, 159 mmol) and
triethylamine (64.4 g, 636 mmol) in anhydrous dichloromethane (600
mL) cooled in an ice bath, under an argon atmosphere. The mixture
was allowed to stir at room temperature for 3 days under an argon
atmosphere. The reaction mixture was extracted with ice-cold 0.5N
hydrochloric acid (1.3 L) and brine (600 mL). The dichloromethane
solution was dried over sodium sulfate, filtered, and concentrated
under reduced pressure. The remaining yellow foam (87.1 g) was
purified by column chromatography on silica gel (1 Kg), eluting
with heptane-ethyl acetate (4:1). The product containing fractions
were combined, concentrated under reduced pressure, and dried under
high vacuum. The amine 4 (40.0 g, 45% yield) was isolated as a
white solid.
[0805] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 13.22 (1H, s,
br), 7.32-7.19 (9H, m), 4.80 (1H, dd, J=12.3, 7.8 Hz), 4.75 (1H, d,
J=16.8 Hz), 4.17 (1H, d, J=16.8 Hz), 3.75 (1H, dd, J=9.0, 3.3 Hz),
3.18 (1H, m), 2.86 (2H, m), 2.66 (1H, m), 2.53 (1H, m), 2.00 (1H,
m), 1.78 (1H, m), 1.60 (1H, m), 1.41 (9H, s), 1.40 (9H, s).
[0806] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 175.15, 171.44,
166.64, 154.36, 141.25, 139.77, 134.87, 129.28, 128.69, 128.41,
128.26, 127.55, 126.03, 122.54, 82.94, 82.59, 57.07, 55.39, 51.76,
34.73, 33.42, 32.95, 28.07, 28.00, 27.85.
4) Preparation of the L-serine Ester of Benazepril:
[0807] Coupling Procedure: ##STR112##
[0808] A mixture of acid 4 (15.5 g, 28.0 mmol), boc-L-serine
tert-butyl ester (6.66 g, 25.5 mmol, prepared by the literature
method), 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (EDCI, 6.84 g, 35.7 mmol), and DMAP (0.436 g, 3.57
mmol) in anhydrous dichloromethane (180 mL) was stirred at room
temperature under an argon atmosphere for 2 days. The solution was
diluted with dichloromethane and washed with water (300 mL), 5%
sodium bicarbonate solution (300 mL), and brine (300 mL). After
drying over sodium sulfate, filtration, and concentration under
reduced pressure, the remaining mixture (22.37 g) was purified by
column chromatography on silica gel (600 g), eluting with heptane
and ethyl acetate (4:1). The product containing fractions (16.38 g)
were combined and concentrated and purified a second time by column
chromatography on silica gel (1 Kg), eluting with heptane and ethyl
acetate (6:1). The product containing fractions were combined,
concentrated, and dried under high vacuum. The protected L-serine
ester of benazepril SPIB0050101 (15.4 g, 76% yield) was isolated as
a white foam.
[0809] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.30-7.10 (9H,
m), 6.22 (1H, d, J=6.9 Hz), 5.36 (1H, d, J=17.4 Hz), 4.98 (1H, m),
4.70 (1H, d, J=9.6 Hz), 4.58 (1H, m), 4.45 (1H, d, J=8.7 Hz), 4.28
(1H, m), 4.04 (1H, d, J=17.4 Hz), 3.32 (1H, m), 2.90 (1H, m),
2.73-2.52 (3H, m), 2.06 (2H, m), 1.77 (1H, m), 1.49 (9H, s), 1.41
(9H, s), 1.34 (18H, s).
[0810] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 171.59, 170.95,
168.83, 167.50, 155.86, 154.14, 141.52, 140.07, 135.70, 129.35,
128.45, 128.31, 127.71, 126.89, 125.86, 122.03, 82.03, 81.89,
81.02, 79.56, 64.89, 57.28, 53.64, 53.28, 49.54, 36.19, 34.80,
34.31, 28.49, 28.32, 27.95.
[0811] HPLC analysis:
[0812] 98.41% purity; r.t.=11.595 min.; 25% DIUF water/75%
acetonitrile; 1 mL/min; 40.degree. C.; Synergi Polar-RP (serial #
161309-2), 4.6.times.250 mm; 20 ul injection. Deprotection and
Purification: ##STR113##
[0813] A solution of hydrogen chloride (1.0 M in acetic acid, 384
mL) was added drop-wise to a solution of the protected L-serine
ester of benazepril SPIB0050101 (15.3 g, 19.2 mmol) in anhydrous
dichloromethane (240 mL) that was cooled in an ice batch, under an
argon atmosphere. The mixture was stored for 7 days at 5.degree. C.
The mixture was concentrated under reduced pressure and dried under
high vacuum to generate a colorless gel. Ethyl acetate (150 mL) was
added to the gel and the mixture was sonicated for 5 minutes. The
white, solid product was concentrated under reduced pressure. An
additional amount of ethyl acetate (150 mL) was added to the solid
and the mixture was heated to reflux for 10 minutes. The solution
was cooled to room temperature. The solids were filtered and dried
under high vacuum. The benazepril-serine ester salt (12.3 g) was
purified by stirring the material in anhydrous dichloromethane (60
mL) overnight, at room temperature. After filtration and drying,
the recovered material (9.3 g) was purified a final time by
stirring the material in ethyl acetate (60 mL) overnight, at room
temperature. The final salt was filtered and dried under high
vacuum (at 45.degree. C.) overnight. In order to remove traces of
solvent the final salt (8.70 g) was dissolved in DIUF water (150
mL) and freeze-dried. Benazeprilat L-serine ester, dihydrochloride
SPIB00501 (8.61 g, 80% yield, 99.69% purity by HPLC) was isolated
in monohydrate form (as white solid).
[0814] .sup.1H NMR (300 MHz, DMSO): .delta. 10.89 (4H, s, br), 8.88
(3H, s, br), 7.33-7.17 (9H, m), 4.67 (1H, d, J=17.4 Hz), 4.47 (3H,
m), 4.28 (1H, m), 3.83 (1H, m), 3.73 (1H, t, J=9.0 Hz), 3.23 (1H,
m), 2.66 (3H, m), 2.52 (1H, m), 2.26 (1H, m), 2.09 (2H, m).
[0815] .sup.13C NMR (75 MHz, DMSO): .delta. 169.76, 168.27, 168.11,
167.68, 140.51, 139.72, 134.67, 129.29, 128.35, 128.24, 127.94,
126.88, 125.96, 123.14, 62.85, 58.33, 56.34, 51.05, 50.29, 33.88,
31.78, 30.54, 26.87.
[0816] HPLC analysis:
[0817] 99.69% purity, r.t.=12.408 min, sample dissolved in DIUF
water/ACN, 82% DIUF water (0.1% TFA)/18% ACN, Synergi Polar-RP
(#258258-4), 4 u, 250.times.4.6 mm, 1 mL/min., 30.degree. C., 20 uL
inj. vol., SPD-10 Avp, chl-210 nm.
[0818] Specific rotation: -123.3 deg (20.degree. C., 21.3 mg/2 mL
ethanol, 589 nm).
[0819] Melting point: 148.0.degree. C. decomposed (Thomas Hoover
CMPA, uncorrected)
[0820] CHN analysis:
[0821] calc.: C, 53.29; H, 5.85; N, 7.46; Cl, 10.70.
(C.sub.25H.sub.29N.sub.3O.sub.7.1.7 HCl--H.sub.2O); found: C,
53.30; H, 5.86; N, 7.35; Cl, 10.78.
5) Preparation of the L-threonine Ester of Benazepril:
[0822] Coupling Procedure: ##STR114##
[0823] A mixture of acid 4 (20.7 g, 37.5 mmol), boc-L-threonine
tert-butyl ester (9.36 g, 34.0 mmol, prepared by the literature
method).sup.1, 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (EDCI, 9.12 g, 47.6 mmol), and DMAP (0.582 g, 4.76
mmol) in anhydrous dichloromethane (250 mL) was stirred at room
temperature under an argon atmosphere for 3 days. The reaction
solution was washed with water (2.times.250 mL), 5% sodium
bicarbonate solution (250 mL), and brine (250 mL). After drying
over sodium sulfate, filtration, and concentration under reduced
pressure, the remaining yellow oil (38.7 g) was purified by column
chromatography on silica gel (1 Kg), eluting with heptane-ethyl
acetate (4:1). The product containing fractions were combined and
concentrated. The L-threonine ester of benazepril SPIB0050201
(18.51 g, 67% yield) was isolated.
[0824] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.26-7.16 (9H,
m), 5.63 (1H, s, br), 5.40 (1H, s, br), 4.99 (2H, m), 4.11 (1H, d,
J=15.6 Hz), 3.27 (1H, s, br), 2.68 (2H, s, br), 2.56 (1H, dd,
J=13.2, 6.0 Hz), 2.35 (2H, s, br), 2.05 (2H, m), 1.47-1.26 (39H,
m).
[0825] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 170.34, 169.72,
168.86, 167.71, 156.18, 154.83, 141.23, 140.60, 135.58, 129.08,
128.41, 127.61, 126.53, 125.91, 122.09, 82.26, 81.68, 80.85, 79.66,
71.64, 58.04, 55.76, 54.23, 50.01, 35.20, 34.54, 33.87, 28.41,
28.28, 27.96, 17.28.
[0826] HPLC analysis:
[0827] 98.30% purity; r.t.=11.924 min.; 25% DIUF water/75%
acetonitrile; 1 mL/min; 40.degree. C.; Synergi Polar-RP (serial #
161309-2), 4.6.times.250 mm; 20 ul injection, 210 nm.
[0828] Melting point: 74.8.degree. C. Deprotection and
Purification: ##STR115##
[0829] A solution of hydrogen chloride (1.0 M in acetic acid, 452
mL) was added drop-wise to a solution of the protected L-threonine
ester of benazepril SPIB0050201 (18.3 g, 22.6 mmol) in anhydrous
dichloromethane (250 mL) that was cooled in an ice batch, under an
argon atmosphere. The reaction mixture was stored for 8 days at
5.degree. C., then concentrated under reduced pressure and dried
under high vacuum to generate white foam. Ethyl acetate (200 mL)
was added to the foam and the mixture was sonicated for 5 minutes.
The white, solid product was concentrated under reduced pressure.
An additional amount of ethyl acetate (200 mL) was added to the
solid and the mixture was heated to reflux for 15 minutes. After
cooling the solution to room temperature, the solids were filtered
and dried under high vacuum (at 40.degree. C.) until a constant
weight was achieved. The remaining salt (11.9 g) was purified by
stirring in anhydrous dichloromethane (90 mL) for three days at
room temperature. The salt was filtered and dried under high vacuum
(at 45.degree. C.) until a constant weight was achieved. In order
to remove traces of solvent the final salt (11.02 g) was suspended
in DIUF water (200 mL) and freeze-dried. The benazeprilat
L-threonine ester, dihydrochloride SPIB00502 (10.87 g, 81% yield,
97.55% purity by HPLC) was isolated in dihydrate form (as a white
solid).
[0830] .sup.1H NMR (300 MHz, DMSO): 810.62 (3H, s, br), 8.85 (4H,
s, br), 7.33-7.18 (9H, m), 5.29 (1H, m), 4.65 (1H, d, J=17.1 Hz),
4.42 (1H, d, J=17.1 Hz), 4.12 (1H, d, J=2.4 Hz), 3.84 (1H, m), 3.58
(1H, dd, J=8.7, 8.1 Hz), 3.23 (1H, m), 2.67 (3H, m), 2.54 (1H, m),
2.26 (1H, m), 2.09 (2H, m), 0.97 (3H, d, J=6.3 Hz).
[0831] .sup.13C NMR (75 MHz, DMSO): .delta. 169.73, 167.81, 167.35,
166.45, 140.28, 139.67, 134.59, 129.33, 128.36, 127.97, 127.04,
126.11, 123.14, 69.75, 58.70, 56.21, 55.40, 50.07, 33.58, 31.97,
30.64, 26.73, 16.09.
[0832] HPLC analysis:
[0833] 97.55% purity, r.t.=15.517 min, sample dissolved in DIUF
water/ACN, 82% DIUF water (0.1% TFA)/18% ACN, Synergi Polar-RP
(#258258-4), 4 u, 250.times.4.6 mm, 1 mL/min., 35.degree. C., 20 uL
inj. vol., SPD-10 Avp, chl-210 nm.
[0834] Specific rotation: -134 deg (21.degree. C., 21.6 mg/2 mL
ethanol, 589 nm)
[0835] Melting point: 152.0-153.0.degree. C. (Thomas Hoover CMPA,
uncorrected)
[0836] CHN analysis:
[0837] calc.: C, 52.59; H, 6.15; N, 7.08; Cl, 10.75.
(C.sub.26H.sub.31N.sub.3O.sub.7.1.8HCl-1.7H.sub.2O); found: C,
52.81; H, 6.15; N, 7.37; Cl, 10.79.
6) Preparation of the L-hydroxyproline Ester of Benazepril:
[0838] Coupling Procedure: ##STR116##
[0839] A mixture of acid 4 (35.8 g, 64.8 mmol),
boc-L-hydroxyproline tert-butyl ester (16.9 g, 58.9 mmol, prepared
by the literature method),
1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI,
15.8 g, 82.5 mmol), and DMAP (1.01 g, 8.25 mmol) in anhydrous
dichloromethane (600 mL) was stirred at room temperature under an
argon atmosphere for 6 days. The dichloromethane solution was
washed with water (400 mL), 5% sodium bicarbonate solution (400
mL), and brine (400 mL). After drying over sodium sulfate,
filtration, and concentration under reduced pressure, the remaining
brown oil (51.5 g) was purified by column chromatography on silica
gel (1 Kg), eluting with heptane-ethyl acetate (4:1). The product
containing fractions were combined and concentrated. The protected
L-hydroxyproline ester of benazepril SPIB0050301 (19.69 g, 41%
yield) was isolated as white foam.
[0840] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.27-7.12 (9H,
m), 5.33 (1H, m), 4.85 (2H, dd, J=17.1, 5.4 Hz), 4.36 (1H, m), 4.23
(1H, t, J=7.50 Hz), 4.10 (1H, dd, J=17.1, 3.6 Hz), 3.73 (1H, m),
3.65 (1H, m), 3.24 (1H, m), 2.71 (2H, m), 2.57-2.40 (3H, m),
2.21-1.97 (4H, m), 1.44 (36H, m), 1.47-1.37 (36H, m).
[0841] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 171.53, 171.42,
170.90, 170.84, 170.59, 167.62, 154.61, 153.90, 153.48, 141.29,
141.25, 140.69, 135.46, 129.13, 128.43, 128.34, 127.71, 126.58,
125.92, 122.20, 81.72, 81.24, 80.78, 80.03, 79.88, 73.04, 72.25,
58.52, 58.04, 54.58, 52.03, 51.67, 50.41, 36.72, 35.58, 34.52,
33.93, 28.40, 28.34, 28.04, 27.99. Deprotection and Purification:
##STR117##
[0842] A solution of hydrogen chloride (1.0 M in acetic acid, 400
mL) was added drop-wise to a solution of the protected
L-hydroxyproline ester of benazepril SPIB0050301 (19.69 g, 24.0
mmol) in anhydrous dichloromethane (265 mL) that was cooled in an
ice batch, under an argon atmosphere. The reaction mixture was
stored for 4 days at 5.degree. C. and then was concentrated under
reduced pressure and dried under high vacuum to generate a light,
yellow foam. Ethyl acetate (200 mL) was added to the yellow foam
and the mixture was sonicated for 5 minutes. The white, solid
product was concentrated under reduced pressure. An additional
amount of ethyl acetate (200 mL) was added to the solid and the
mixture was heated to reflux for 15 minutes. The solution was
cooled to room temperature. The solids were filtered and dried
under high vacuum at 35.degree. C. The remaining solid (14.50 g)
was purified by stirring in anhydrous dichloromethane (60 mL)
overnight at room temperature. The remaining salt (13.19 g) was
filtered and dried under high vacuum at 45.degree. C. for 20 hours.
In order to remove trace of solvent, the salt was dispersed in DIUF
water (500 mL) and freeze-dried. Benazeprilat
trans-4-hydroxy-L-proline ester, dihydrochloride SPIB00503 (12.5 g,
87% yield, 99.95% purity by HPLC) was isolated in monohydrate form
(as a white solid).
[0843] .sup.1H NMR (300 MHz, DMSO): .delta. 10.34 (6H, s, br),
7.34-7.17(9H, m), 5.25 (1H, m), 4.66 (1H, d, J=17.4 Hz), 4.43 (1H,
d, J=17.4 Hz), 4.24 (1H, t, J=9.0 Hz), 3.88 (1H, m), 3.77 (1H, t,
J=9.0 Hz), 3.51 (1H, dd, J=12.6, 3.9 Hz), 3.23 (2H, m), 2.66 (4H,
m), 2.22 (3H, m), 2.11 (2H, m).
[0844] .sup.13C NMR (75 MHz, DMSO): .delta. 169.80, 169.01, 167.93,
167.88, 140.40, 139.76, 134.59, 129.17, 128.33, 128.27, 127.87,
126.87, 126.01, 123.13, 74.10, 57.88, 57.39, 56.31, 50.23, 49.95,
33.98, 33.69, 31.68, 30.60, 26.79.
[0845] HPLC analysis:
[0846] 99.95% purity, r.t.=16.725 min, sample dissolved in
DIUF/ACN, 82% DIUF water (0.1% TFA)/18% ACN, Synergi Polar-RP
(#258258-4), 4 u, 250.times.4.6 mm, 1 mL/min., 30.degree. C., 20 uL
inj. vol., SPD-10 Avp, chl-210 nm.
[0847] Specific rotation: -116.0 deg (20.degree. C., 23.7 mg/2 mL
ethanol, 589 nm)
[0848] Melting point: 168.0.degree. C. decomposed (Thomas Hoover
CMPA, uncorrected)
[0849] CHN analysis:
[0850] calc.: C, 55.52; H, 5.96; N, 7.19; Cl, 9.41.
(C.sub.27H.sub.31N.sub.3O.sub.7.1.55HCl--H.sub.2O); found: C,
55.55; H, 5.99; N, 7.16; Cl, 9.67.
[0851] Literature Cited: Moore, J. W.; Szelke, M. Tetrahedron Lett.
1970, 4423-4426.
Synthesis and Efficacy Studies of Enalapril Drivatives:
Overview:
[0852] The procedure for the synthesis of the L-serine,
L-threonine, and L-hydroxyproline esters of enalapril is outlined
in Synthetic Sequence section. The complete procedure and
analytical data are given in the Experimental Section. In general,
the maleate salt of enalapril (Xiangding Chemical International
Company) was butylated to protect the free acid group. The ethyl
ester was then hydrolyzed to the acid with sodium hydroxide,
followed by acidification. The amine present in the molecule was
protected with a tert-butyloxycarbonyl group to prevent
dimerization during the subsequent coupling reaction. The protected
intermediate was then coupled with the tert-butyl ester of
boc-L-serine, boc-L-threonine, or boc-L-hydroxyproline using EDC.
The protected intermediate esters were treated with dilute
hydrochloric acid in acetic acid at low temperature to remove all
of the protecting groups. Washing with ethyl acetate and
dichloromethane (or ACN) purified the amino acid ester salts of
Enalapril. The final salts were dried under high vacuum and
analyzed for NMR, HPLC, CHN, specific rotation, and melting point.
Synthetic Sequence: ##STR118## ##STR119## Synthesis of the
L-serine, L-threonine, and L-hydroxyproline Esters of Enalapril: a)
THF; b) NaOH, MeOH, H.sub.2O; c) (Boc).sub.2O, Et.sub.3N,
CH.sub.2Cl.sub.2; d) Boc-Ser-OtBu, EDC, DMAP, CH.sub.2Cl.sub.2; e)
Boc-Thr-OtBu, EDC, DMAP, CH.sub.2Cl.sub.2; f) Boc-Hyp-OtBu, EDC,
DMAP, CH.sub.2Cl.sub.2; g) HCl, AcOH, CH.sub.2Cl.sub.2.
Experimental Section:
[0853] The synthesis of SPIB00401, SPIB00402 and SPIB00403 was
conducted in batches. Reagents mentioned in the experimental
section were purchased at the highest obtainable purity from
Lancaster, Sigma-Aldrich, Acros, Bachem, or Xiangding Chemical
International, except for solvents, which were purchased from
either Fisher Scientific or Mallinkrodt. 1) Butylation of
Enalapril: ##STR120##
[0854] A solution of 2-tert-butyl-1,3-dicyclohexyl isourea 1 (592
g, 2.11 mol, prepared by the literature procedure) in THF (500 mL)
was added drop-wise to a mixture of enalapril maleate (130 g, 0.264
mol) in anhydrous THF (600 mL) cooled in an ice-water bath. The
mixture was allowed to stir for 24 hours at room temperature under
an argon atmosphere. The reaction mixture was filtered, and the
precipitate (DCU) was washed with dichloromethane (3.times.500 mL).
The filtrates were combined and concentrated to produce a green
oil. The residue was diluted with dichloromethane (1 L), washed
with 5% sodium bicarbonate solution (1 L), and washed with brine
(500 mL). The dichloromethane solution was dried over sodium
sulfate, filtered, and concentrated under reduced pressure. The
remaining mixture (189 g) was purified by column chromatography on
silica gel (1 Kg), eluting with heptane-ethyl acetate (2:1). The
product containing fractions were combined and concentrated under
reduced pressure and dried at high vacuum. Butylated enalapril 2
(87.3 g, 76% yield) was obtained as a light yellow oil.
[0855] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.31-7.15 (5H,
m), 4.43 (1H, dd, J=8.4, 3.3 Hz), 4.18 (2H, q, J=7.2 Hz), 3.52 (3H,
m), 3.25 (1H, t, J=6.6 Hz), 2.67 (2H, m), 2.24 (1H, s, br), 2.18
(1H, m), 1.98 (5H, m), 1.45 (9H, s), 1.29 (3H, d, J=7.2 Hz), 1.25
(3H, t, J=7.2 Hz).
[0856] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 173.99, 173.03,
170.81, 141.07, 128.15, 128.09, 125.68, 81.03, 60.59, 59.93, 59.44,
53.36, 46.41, 35.06, 31.95, 28.86, 27.88, 24.72, 18.80, 14.28. 2)
Saponification of the Butylated Enalapril: ##STR121##
[0857] A sodium hydroxide solution (80.8 g, 2.02 mol) in DIUF water
(1 L) was added in a drop-wise fashion to the butylated enalapril 2
(87.2 g, 0.202 mol) dissolved in methanol (1 L), while cooling the
methanol solution in an ice bath. The solution was allowed to stir
for 18 hours under an argon atmosphere while cooling in an ice
bath. The reaction mixture was concentrated under reduced pressure
and dried under high vacuum to produce a cloudy solution (847 g).
The residue was cooled in an ice bath and a solution of acetic acid
in water (1.2 L, 1:10 v/v acetic acid/water) was added drop-wise to
adjust the solution to pH=7. A white precipitate formed which was
extracted with dichloromethane (4.times.500 mL). The combined
dichloromethane fractions were dried over sodium sulfate, filtered,
and concentrated under reduced pressure to generate a brown
foam-like solid (73.4 g). After additional drying under high
vacuum, 3 (61.5 g, 75% yield) was obtained as a light brown solid,
which was used without additional purification.
[0858] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.12 (5H, m),
5.31 (2H, s, br), 4.41 (1H, dd, J=9.0, 3.6 Hz), 3.97 (1H, q, J=6.6
Hz), 3.53 (1H, m), 3.42 (1H, m), 3.31 (1H, t, J=6.3 Hz), 2.72 (2H,
m), 2.21-1.91 (6H, m), 1.52 (3H, d, J=6.6 Hz), 1.43 (9H, s).
[0859] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 172.66, 170.29,
168.74, 140.46, 128.30, 128.18, 125.80, 81.63, 61.74, 59.75, 54.57,
46.64, 35.53, 31.89, 28.90, 27.97, 24.61, 16.47. 3) Amine
Protection: ##STR122##
[0860] A solution of di-tert-butyl dicarbonate (36.5 g, 167 mmol)
in anhydrous dichloromethane (200 mL) was added in a drop-wise
fashion to a solution of 3 (61.5 g, 152 mmol) and triethylamine
(61.5 g, 608 mmol) in anhydrous dichloromethane (600 mL) cooled in
an ice bath, under an argon atmosphere. The mixture was allowed to
stir at room temperature for 3 days at room temperature under an
argon atmosphere. The reaction mixture was washed with ice-cold
0.5N hydrochloric acid (1.2 L) and brine (600 mL). The
dichloromethane solution was dried over sodium sulfate, filtered,
and concentrated under reduced pressure. The remaining yellow oil
(87 g) was purified by column chromatography on silica gel (1 Kg),
eluting with heptane-ethyl acetate (3:1). The product containing
fractions were combined, concentrated under reduced pressure, and
dried under high vacuum until the weight was constant. A protected
amine 4 (28.7 g, 37% yield) was obtained as a white solid.
[0861] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 13.61 (1H, br s),
7.30-7.16 (5H, m), 4.79 (1H, q, J=7.5 Hz), 4.47 (1H, dd, J=8.7, 4.5
Hz), 3.75 (2H, m), 3.54 (1H, m), 2.84 (2H, m), 2.68 (1H, m), 2.22
(1H, m), 1.98 (4H, m), 1.43 (9H, s), 1.10 (3H, d, J=7.5 Hz).
[0862] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 174.62, 172.12,
170.10, 154.70, 141.23, 128.57, 128.41, 126.00, 82.74, 81.81,
60.00, 55.63, 53.00, 46.89, 34.27, 33.40, 28.80, 28.16, 27.99,
24.86, 14.08.
4) Preparation of the L-serine Ester of Enalapril:
[0863] Coupling Procedure: ##STR123##
[0864] A mixture of 4 (15.3 g, 30.3 mmol), boc-L-serine tert-butyl
ester (7.19 g, 27.5 mmol, prepared by the literature method),
1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI,
7.38 g, 38.5 mmol), and DMAP (0.47 g, 3.85 mmol) in anhydrous
dichloromethane (200 mL) was stirred at room temperature under an
argon atmosphere for 4 days. The dichloromethane solution was
washed with water (2.times.200 mL) and brine (200 mL). After drying
the dichloromethane solution over sodium sulfate, filtration, and
concentration under reduced pressure, the residue (24.6 g) was
purified by column chromatography on silica gel (700 g), eluting
with heptane-ethyl acetate (4:1). The product containing fractions
were combined and concentrated. The remaining white solid (14.1 g)
was crystallized from heptane (100 mL) and methyl tert-butyl ether
(50 mL). After filtration and drying, the protected L-serine ester
of enalapril SPIB0040101 (13.5 g, 66% yield) was obtained as a
white solid.
[0865] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.29 (2H, m),
7.20 (3H, m), 5.55 (1H, d, J=7.8 Hz), 5.26 (1H, m), 4.50-4.23 (5H,
m), 3.79 (1H, m), 3.66 (1H, m), 2.76 (1H, m), 2.58 (2H, m), 2.25
(1H, m), 1.97 (4H, m), 1.44 (36H, m), 1.27 (3H, d, J=6.6 Hz).
[0866] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 171.11, 169.36,
168.33, 155.18, 154.32, 141.43, 128.21, 128.19, 125.78, 82.48,
80.98, 80.86, 79.75, 65.05, 59.58, 55.70, 53.34, 50.12, 47.00,
35.20, 34.19, 29.14, 28.35, 28.27, 27.96, 27.90, 24.50, 16.55.
[0867] HPLC analysis:
[0868] 98.81% purity; r.t.=9.433 min.; 25% DIUF water/75%
acetonitrile; 1 mL/min; 39.degree. C.; Synergi Polar-RP (serial #
234257-1), 4.6.times.250 mm; 20 ul injection; RI 210 nm.
[0869] Melting point: 134-135.degree. C. (Thomas Hoover CMPA,
uncorrected) Deprotection and Purification: ##STR124##
[0870] A solution of hydrogen chloride (1.0 M in acetic acid, 360
mL) was added drop-wise to a solution of the protected L-serine
ester of enalapril SPIB0040101 (13.4 g, 17.9 mmol) in anhydrous
dichloromethane (190 mL) that was cooled in an ice batch, under an
argon atmosphere. The mixture was stored for 8 days at 5.degree. C.
The mixture was concentrated under reduced pressure and dried under
high vacuum to generate a light, yellow oil (19.56 g). Ethyl
acetate (100 mL) was added to the oil and the mixture was sonicated
for 5 minutes. The white, solid product (14.8 g) was filtered and
dried under high vacuum. An additional amount of ethyl acetate (200
mL) was added to the solid and the mixture was heated to reflux for
10 minutes. While the solution was cooling, the solid was sonicated
for 30 minutes and stirred for 2 hours at room temperature. The
solids were filtered and dried under high vacuum. The remaining
salt (10.5 g) was purified twice by stirring the material in
anhydrous dichloromethane (60 mL) overnight, at room temperature.
The final salt was filtered and dried under high vacuum at room
temperature until a constant weight was obtained. Enalaprilic acid
L-serine ester, dihydrochloride SPIB00401 (8.01 g, 88% yield,
97.09% purity by HPLC) was obtained in monohydrate form (as white
solid).
[0871] .sup.1H NMR (300 MHz, DMSO): .delta. 10.76 (4H, s, br), 9.02
(3H, s, br), 7.25 (5H, m), 4.67 (2H, m), 4.35 (3H, m), 3.93 (1H,
m), 3.70 (1H, m), 3.42 (1H, m), 2.62 (2H, m), 2.17 (3H, m), 1.89
(3H, m), 1.44 (3H, d, J=6.0 Hz).
[0872] .sup.13C NMR (75 MHz, DMSO): .delta. 172.53, 167.89, 167.03,
166.94, 140.26, 128.33, 126.11, 63.21, 58.90, 58.44, 53.97, 51.13,
46.83, 31.44, 30.45, 28.65, 24.71, 15.35.
[0873] HPLC analysis:
[0874] 97.09% purity; r.t.=8.367 min.; 85% DIUF water (0.1%
TFA)/15% ACN; 1 mL/min; 36.0 C; Synergi Polar-RP 5 u column (serial
# 161309-2), 4.6.times.250 mm; 20 ul injection; Sig=210.4.
[0875] HRMS [M-2HCl--H.sub.2O+H]: calc. 436.2084
(C.sub.21H.sub.31Cl.sub.2N.sub.3O.sub.7); found 436.2079.
[0876] Melting point: 129.5.degree. C. decomposed (Thomas Hoover
CMPA, uncorrected)
[0877] CHN analysis:
[0878] calc.: C, 47.91; H, 6.32; N, 7.98.
(C.sub.21H.sub.31Cl.sub.2N.sub.3O.sub.7--H.sub.2O); found: C,
47.95; H, 6.32; N, 7.83.
5) Preparation of the L-threonine Ester of Enalapril:
[0879] Coupling Procedure: ##STR125##
[0880] A mixture of 4 (28.4 g, 56.3 mmol), boc-L-threonine
tert-butyl ester (14.1 g, 51.2 mmol, prepared by the literature
method), 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (EDCI, 13.7 g, 71.7 mmol), and DMAP (0.876 g, 7.17
mmol) in anhydrous dichloromethane (450 mL) was stirred at room
temperature under an argon atmosphere for 13 days. The
dichloromethane solution was washed with water (2.times.500 mL) and
brine (500 mL). After drying over sodium sulfate, filtration, and
concentration under reduced pressure, the remaining yellow oil
(46.49 g) was purified by column chromatography on silica gel (1
Kg), eluting with heptane-ethyl acetate (5:1). The product
containing fractions were combined in two fractions and
concentrated. Protected L-threonine ester of enalapril SPIB0040201
(7.35 g and 6.83 g, 36.3% yield) was present in both fractions.
Both fractions were deprotected as described below and recombined
in the final purification.
[0881] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.30 (2H, m),
7.21 (3H, m), 5.28 (2H, m), 5.14 (1H, d, J=8.4 Hz), 4.30-4.22 (3H,
m), 3.85 (1H, m), 3.65 (1H, m), 2.65 (2H, m), 2.39 (1H, m), 2.20
(1H, m), 1.99 (4H, m), 1.46 (36H, m), 1.25 (6H, m).
[0882] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 171.23, 170.89,
170.03, 168.57, 155.82, 154.64, 141.43, 128.37, 128.24, 125.92,
82.83, 81.06, 80.01, 79.73, 72.23, 68.34, 59.80, 59.20, 57.69,
55.65, 50.78, 47.08, 34.40, 29.14, 28.41, 28.30, 28.03, 24.67,
20.01, 17.15. Deprotection and Purification: ##STR126##
[0883] A solution of hydrogen chloride (1.0 M in acetic acid, 185
mL) was added drop-wise to a solution of one of the batches of the
protected L-threonine ester of enalapril SPIB0040261 (7.05 g, 9.25
mmol batch 1, 6.63 g, 8.70 mmol, batch 2) in anhydrous
dichloromethane (100 mL) that was cooled in an ice batch, under an
argon atmosphere. The mixture was stored for 7 days at 5.degree. C.
The mixture was concentrated under reduced pressure and dried under
high vacuum to generate a colorless gel. Ethyl acetate (250 mL) was
added to the gel and the mixture was sonicated for 5 minutes. The
white, solid product was filtered and dried under high vacuum. An
additional amount of ethyl acetate (100 mL) was added to the solid
and the mixture was heated to reflux for 10 minutes. After cooling
the solution to room temperature, the solids were filtered and
dried under high vacuum until a constant weight was achieved. The
deprotection step was then repeated with the second batch of
material.
[0884] Both batches of the final salt (4.84 g from batch 1 and 4.30
from batch 2) were combined and purified by stirring in anhydrous
dichloromethane (60 mL) overnight at room temperature. The final
salt was filtered and dried under high vacuum. The washing
procedure was then repeated for two days with ethyl acetate (40 mL)
and for 18 hours with acetonitrile (50 mL). After filtration and
drying, enalaprilic acid L-threonine ester, dihydrochloride
SPIB00402 (7.75 g, 82% yield, 97.61% purity by HPLC) was isolated
in monohydrate form (as white solid).
[0885] .sup.1H NMR (300 MHz, DMSO): .delta. 10.50 (3H, s, br), 8.96
(4H, s, br), 7.24 (5H, m), 5.44 (1H, m), 4.58 (0.3H, m), 4.34-3.90
(3.4H, m), 3.78 (0.3H, m), 3.64 (0.7H, m), 3.47 (1H, m), 3.29
(0.3H, m), 2.72 (1H, m), 2.52 (1H, m), 2.16 (3H, m), 1.87 (2.7H,
m), 1.60 (0.3H, m), 1.44 (6H, m).
[0886] .sup.13C NMR (75 MHz, DMSO): .delta. 173.21, 172.55, 168.02,
167.88, 166.99, 166.71, 140.36, 128.37, 126.14, 70.43, 70.22,
58.96, 58.25, 58.13, 55.56, 54.85, 53.63, 46.65, 46.48, 32.13,
31.55, 30.47, 28.67, 24.70, 21.88, 16.99, 16.56, 16.50, 15.12.
[0887] HPLC analysis:
[0888] 97.61% purity, r.t.=9.258 min, sample dissolved in DIUF
water/ACN, 85% DIUF water (0.1% TFA)/15% ACN, Synergi Polar-RP
(#161309-2), 4 u, 250.times.4.6 mm, 1 mL/min., 40.degree. C., 20 uL
inj. vol., SPD-10 Avp, chl-210 nm.
[0889] Specific rotation: -33.8 deg (21.degree. C., 17.3 mg/2 mL
ethanol, 589 nm)
[0890] Melting point: 147.0-148.0.degree. C. decomposed (Thomas
Hoover CMPA, uncorrected)
[0891] CHN analysis:
[0892] calc.: C, 49.90; H, 6.61; N, 7.94; Cl, 11.38.
(C.sub.22H.sub.31N.sub.3O.sub.7.1.7HCl--H.sub.2O); found: C, 50.12;
H, 6.61; N, 7.90; Cl, 11.33.
6) Preparation of the L-hydroxyproline Ester of Enalapril:
[0893] Coupling Procedure: ##STR127##
[0894] A mixture of 4 (24.6 g, 48.7 mmol), boc-L-hydroxyproline
tert-butyl ester (12.7 g, 44.3 mmol, prepared by the literature
method), 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (EDCI, 11.9 g, 62.0 mmol), and DMAP (0.757 g, 6.20
mmol) in anhydrous dichloromethane (400 mL) was stirred at room
temperature under an argon atmosphere for 9 days. The
dichloromethane solution was washed with water (400 mL), 5% sodium
bicarbonate solution (400 mL), water (400 mL), and brine (400 mL).
After drying the dichloromethane solution over sodium sulfate,
filtration, and concentration under reduced pressure, the residual
green oil (42.0 g) was purified by column chromatography on silica
gel (1 Kg), eluting with heptane-ethyl acetate (5:1). The product
containing fractions were combined, concentrated, and dried under
high vacuum till a constant weight was obtained, yielding protected
L-hydroxyproline ester of enalapril SPIB0040301 (11.41 g, 33%
yield).
[0895] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.29 (2H, m),
7.19 (3H, m), 5.24 (2H, m), 4.21 (3H, m), 3.74 (3H, m), 3.50 (1H,
m), 2.77 (1H, m), 2.67-2.50 (2H, m), 2.23 (3H, m), 1.98 (4H, m),
1.44 (36H, m), 1.30 (3H, m).
[0896] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 171.09, 170.91,
169.08, 154.37, 153.80, 153.44, 141.49, 128.37, 128.27, 125.91,
81.48, 81.30, 81.10, 80.32, 73.15, 72.46, 59.78, 58.49, 55.48,
51.95, 51.65, 50.36, 47.27, 36.74, 35.60, 35.17, 34.32, 29.29,
28.40, 28.31, 28.03, 24.70, 16.45. Deprotection and Purification:
##STR128##
[0897] A solution of hydrogen chloride (1.0 M in acetic acid, 290
mL) was added drop-wise to a solution of the protected
L-hydroxyproline ester of enalapril SPIB0040301 (11.23 g, 14.5
mmol) in anhydrous dichloromethane (160 mL) that was cooled in an
ice batch, under an argon atmosphere. The mixture was stored for 8
days at 5.degree. C. The mixture was concentrated under reduced
pressure and dried under high vacuum to generate yellow foam. Ethyl
acetate (100 mL) was added to the yellow foam, and the mixture was
sonicated for 5 minutes. The white, solid product was filtered and
dried under high vacuum. An additional amount of ethyl acetate (100
mL) was added to the solid, and the mixture was heated to reflux
for 10 minutes. After cooling the solution to room temperature, the
solids were filtered and dried under high vacuum until a constant
weight was achieved. The remaining solid (6.89 g) was purified by
stirring in anhydrous dichloromethane (60 mL) overnight at room
temperature. The remaining salt (5.80 g) was filtered and dried
under high vacuum. In order to remove trace of solvent, the salt
was dissolved in water (100 mL) and freeze-dried. Enalaprilic acid
trans-4-hydroxy-L-proline ester, dihydrochloride SPIB00403 (5.40 g,
67% yield, 99.68% purity by HPLC) was obtained in monohydrate form
(as white solid).
[0898] .sup.1H NMR (300 MHz, DMSO): .delta. 10.65 (6H, s, br),
7.32-7.19 (5H, m), 5.44 (0.3H, m), 5.36 (0.7H, m), 4.67 (0.3H, m),
4.44 (1H, t, J=8.7 Hz), 4.28 (1.7H, m), 3.96 (1H, m), 3.73-3.31
(4H, m), 2.78 (1H, m), 2.61 (1H, m), 2.33 (1H, m), 2.18 (3.3H, m),
1.93-1.59 (3.7H, m), 1.46 (3H, m).
[0899] .sup.13C NMR (75 MHz, DMSO): .delta. 173.31, 172.50, 169.24,
167.85, 167.66, 167.21, 140.30, 140.22, 128.27, 128.22, 126.01,
74.64, 58.89, 58.70, 57.92, 57.74, 57.58, 57.39, 54.68, 53.96,
50.31, 50.09, 46.59, 46.41, 34.16, 34.06, 31.93, 31.34, 30.34,
28.55, 24.62, 21.84, 16.53, 15.20.
[0900] HPLC analysis:
[0901] 99.68% purity, r.t.=6.700 min, sample dissolved in DIUF
water/ACN, 82% DIUF water (0.1% TFA)/18% ACN, Synergi Polar-RP
(#258258-4), 4 u, 250.times.4.6 mm, 1 mL/min., 35.degree. C., 20 uL
inj. vol., SPD-10 Avp, chl-210 nm.
[0902] Specific rotation: -36.2 deg (21.degree. C., 16.8 mg/2 mL
ethanol, 589 nm)
[0903] Melting point: 159.0-160.0.degree. C. (Thomas Hoover CMPA,
uncorrected)
[0904] CHN analysis:
[0905] calc.: C, 50.01; H, 6.47; N, 7.61; Cl, 11.87.
(C.sub.23H.sub.31N.sub.3O.sub.7.1.85HCl-1.3H.sub.2O); found: C,
50.11; H, 6.57; N, 7.47; Cl, 11.84.
Effect of Various ACE Inhibitor Enalapril Derivatives on
Hemodynamics in Spontaneously Hypertensive Rats (SHRs).
[0906] ACE inhibitors have been known to be useful in
antihypertensive therapy for many years. Some ACE inhibitors are
delivered orally as the parent drug (captopril) while others are
delivered as pro-drugs in order to obtain clinically useful plasma
concentrations. This study provides data on the effect of these
various enalapril derivatives on hemodynamics in SHRs when given
orally and compared to enalapril.
Methods
[0907] Briefly, SHRs fitted with right carotid catheters were
orally gavaged with 100 mg/kg enalapril or equivalent does of the
enalapril derivatives, and the blood pressures were followed
continuously up to 3 hours and then a measurement at 24 hours. The
animals were conscious throughout the procedure and were placed in
a restrainer and hooked up to a pressure transducer and a signal
processor. After 3 hours, the animals were placed back into their
cage, and blood pressure was finally measured again at 24
hours.
Results
[0908] Enalapril reduced blood pressure at 3 hours and 24 hours by
approximately 20% depending on if data from systolic, diastolic or
mean blood pressure were analyzed. The pressures generally stayed
at baseline levels up to the 3 hour time point, and the pressure at
3 hours was remarkably similar to that seen at 24 hours. Vehicle
treated animals showed no change in blood pressure throughout the
study.
[0909] The reduction in blood pressures caused by the various
agents was associated with a slightly greater drop in diastolic
pressure than in systolic pressure. For the purpose of comparison,
mean arterial blood pressures are utilized for this project
summary. For the enalapril series, enalalpril and the amino acid
derivatives of enalapril reduced arterial blood pressure.
[0910] No reflex tachycardia was noted and this is not unusual for
ACE inhibitors. Heart rates tended to come down with time in all
groups as the animals became more accustomed to the
restrainers.
VII Water Soluble Derivatives of Fibric Acid Derivatives
[0911] Fibric acid compounds are useful anti-hyperlipidemic drugs
useful in the treatment of hyperlipidemia in mammals where the
symptoms are elevated triglycerides, low HDL (High density
lipoproteins or "good" cholesterol, and elevated cholesterol.
Fibric Acid compounds are also useful in reducing LDL (Low density
lipoproteins, or "bad" cholesterol). The general structure of the
fibric acid analogs is represented below, where X is various mixed
aliphatic and aromatic functionalities. Specific compounds included
in this formula are clofibric acid, fenofibric acid, ciprfibrate
and gemfibrozil and the like. ##STR129##
[0912] Typical examples of the chemical moiety X in the above
structure are shown below. ##STR130##
[0913] As used herein, the term "fibric acid compounds" refers to
the fabric acid analogs depicted here which are not bonded to an
amino acid. When the fibric acid compounds are bonded to an amino
acid, they will be referred to as fibric acid "amino acid
derivatives" or like term.
[0914] Fibric acid analogs shown in the structure above have been
shown to have a large number of therapeutic applications, which are
quite diverse and somewhat surprising. Broadly, these compounds are
useful in the treatment dyslipidemia and dyslipoproteinemia.
Dyslipidemia and dyslipoproteinemia are herein defined to include
the group selected from hypercholesterolemia, abnormal and elevated
levels of cholesterol, abnormal and elevated levels of LDL
cholesterol, abnormal and elevated levels of total cholesterol,
abnormal and elevated levels of plasma cholesterol, abnormal and
elevated levels of triglycerides, hypertrigylceridaemia, abnormal
levels of lipoproteins, abnormal and elevated levels of low density
lipoproteins (LDLs), abnormal and elevated levels of very low
density lipoproteins, abnormal and elevated levels of very low
intermediate density lipoproteins, abnormal levels of high density
lipoproteins, hyperlipidemia, hyperchylomicronemia, abnormal levels
of chylomicrons, related disorders, and combinations thereof such
as those described in The ILIB Lipid Handbook for Clinical
Practice, Blood Lipids and Coronary Heart Disease, Second Edition,
A. M. Gotto et al, International Lipid Information Bureau, New
York, N.Y., 2000, the contents of which is hereby incorporated by
reference.
Mechanism of Action:
[0915] The mechanism of action of Fibric acid compounds seen in
clinical practice have been explained in-vivo in transgenic mice
and in vitro in human hepatocytes cultures by the activation of
peroxisome proliferator activated receptor alpha (PPAR-alpha).
Through this mechanism, Fibric acid compounds increase lipolysis
and elimination of triglyceride-rich particles from plasma by
activating lipoprotein lipase and reducing production of apoprotein
C-III (an inhibitor of lipoprotein lipase activity).
[0916] The resulting fall in triglycerides produces an alteration
in the size and composition of LDL from small, dense particles
(which are thought to be atherogenic due their susceptibility to
oxidation), to large buoyant particles. These larger particles have
greater affinity for cholesterol receptors and are catabolized
rapidly. Activation of PPAR-alpha also induces an increase in the
synthesis of apoproteins A-I, A-II, and HDL cholesterol. The Fibric
Acid compounds depicted hereinabove are also useful in the
treatment of gout, as they reduce serum uric acid levels in
hyperurecemic patients.
[0917] Hyperlipidemia types include type I, type IIa, type IIb,
type III, type IV, and type V. These types can be characterized
according to the levels relative to normal levels of lipids
(cholesterol and triglycerides) and lipoproteins in patients.
Different classifications are derived from Drug Facts and
Comparisons, 52nd Edition (1998) page 1066 which is hereby
incorporated by reference.
[0918] Many of the fibric acid compounds when administered orally
do not have sufficient bioavilability, and furthermore absorption
is variable and erratic and is dependent upon the intake of food.
In fact absolute bioavialability of many of the fibric acid
compounds is not possible since the ones currently marketed are
insoluble in water, hence a parenteral formuation is difficult to
prepare or not available. Furthermore, since these drugs usually
are administered as esters, they have to be metabolized in the body
to release active drug, which are the fibric acids. However, due to
the ester formation of these drugs, they are quite insoluble in
water, hence are difficult to formulate, and are not easily broken
down in the body to release active drugs.
[0919] Many of the Fibric acid compounds are low to medium
molecular weight solids with characteristic odor. Taken orally they
have unpleasant taste and can severely irritate mouth and throat.
Taken with food provides more blood concentration compared to
fasting. Overall bioavailability has been reported anywhere between
40-60 and quite variable among patients. As shown hereinbelow, this
fed fast difference in bioavailability is more pronounced when the
Fibric acid compounds are compared with that of the amino acid
derivatives of Fibric acids.
[0920] One of the significant problems associated with currently
marketed fibric acid compounds is that they are administered as
prodrugs to patients which are metabolized to cleave off the
prodrug moiety, e.g., esters or alcohol and the cleaved products
may themselves be highly toxic. For example, in the case of
fenofibrate and gemfibrozil, isopropyl alcohol is released as the
esterase enzyme cleave the pro-moiety from the fenofibric acid. It
is well known that isopropanol is highly toxic when released into
any of the mammalian tissues.
[0921] In order to improve the therapeutic effectiveness, uniform
blood profile, develop pharmaceutically elegant formulation and
improve the solubility of the drug in water, the present invention
provides alternative derivatives of Fibric acid compounds which
overcome many of the difficulties stated above.
[0922] Accordingly, in one aspect, the present invention is
directed to alternate class of derivatives of Fibric acid
compounds, namely amino acid derivatives. The amino acid derivative
consists of the hydroxyl group of an amino acid esterified to the
free carboxyl group present on the Fibric acid compounds. In
another embodiment, the amine group of the amino acid is reacted
with COOH of the fibric acids to form an amide linkage.
[0923] More specifically, in one aspect of the present invention is
directed to, the compounds of the formulas ##STR131## where x is as
defined hereinabove or pharmaceutically acceptable salts thereof;
wherein R is either NH-AA or O-AA and AA is an amino acid, without
the amino group or hydroxy group, in which either an amine group or
the hydroxyl group, respectively, is reacted with the carboxylic
acid group of Fibric acid compounds.
[0924] The present invention is also directed to a pharmaceutical
composition comprising a therapeutically effective amount of the
various Fibric acid amino acid derivatives and a pharmaceutical
carrier therefor.
[0925] In another embodiment, the present invention is directed to
a method of treating a patient in need of Fibric acid therapy,
which method comprises administering to said patient an effective
amount of the Fibric acid amino acid derivatives.
[0926] In a further embodiment, the present invention is directed
to a method of converting liquid Fibric acid compounds into a solid
powder by reacting the carboxyl functionality of the Fibric acid
compounds with either amine or hydroxyl functionality of an amino
acid undo conditions sufficient to form a covalent bond between the
amino acid and the fibric acid compound and isolating the product
thereof.
[0927] In a still further embodiment, the present invention is
directed to a method of substantially and in a therapeutically
efficacious manner, administer the fibric acid amino acid
derivatives which in this form facilites the absorption of the
fibric acid moiety, thereby improving the consistent therapeutic
effect which method comprises administering to a patient the amino
acid derivative of fibric acid, prepared by a process which
comprises reacting the COOH functionality of the Fibric acid
compound with an amino acid, especially either NH.sub.2 of the
amino acid or OH functionality of the hydroxy containing amino
acids to form an amide or ester covalent bond respectively and
isolating the product thereof.
[0928] The present inventor has found that amino acid esterified to
Fibric acid compounds, especially the naturally occurring amino
acids, are pharmaceutically elegant free flowing powders, and are
rapidly absorbed into the body. If cleaved in the body, they
release non-toxic amino acids upon cleavage in the body. However,
the amino acid derivatives of fibric acid have the same utility as
the fibric acid compounds from which they are prepared and which
are not bound to an amino acid. They require none of the
emulsifiers, additives and other excipients.
[0929] Furthermore, the present inventor has found that the amino
acid derivatives of Fibric acid derivatives are highly effective
anti-hyperlipidemics and exhibit such effect intact. The amino acid
derivatives are effective anti-hyperlipidemics and are useful in
the treatment of a number of high cholesterol related illnesses and
exhibit such potential with or without releasing the active parent
drug.
[0930] While the amino acid derivatives of fibric acid of the
present invention are not expected to possess any acidic activity
due to blockage of the carboxylic acid group responsible for such,
the present inventor has found that the amino derivatives of fibric
acid are effective anti-hyperlipidemics with or without releasing
Fibric acid derivatives. However, without wishing to be bound, it
is believed that Fibric acid amino acid derivatives described
herein may or may not release in vivo the active drug with all its
pharmacological and cholesterol lowering properties.
[0931] The present invention clearly provides a number of
advantages over Fibric acid compounds, for example, all of the side
chains cleaved in vivo, if at all, from these derivatives are
naturally occurring essential amino acids and hence are non-toxic.
This results in high therapeutic index. Secondly the amino acid
derivatives may be readily cleaved in the body to release Fibric
acid or its active component. On the other hand, the fibric acid
amino acid derivatives of the present invention exhibit the same
therapeutic properties as the fibric acid compounds from which they
are found thereon. Furthermore, due to their high water solubility,
the amino acid derivatives can be easily administered by either
forming an in-situ solution just before IV administration using
lyophilized sterile powder or providing the drug in solution in
prefilled syringe or bottles for infusion. The amino acid esters
are more stable than Fibric acid compounds since the COOH group in
Fibric acid is blocked to reaction with bases. Thus the Fibric acid
amino acid derivatives described here are more effective then
Fibric acid derivatives itself without the toxicity and other
pharmaceutical problems associated with current marketed
formulations.
[0932] The amino acid derivatives of this invention are
anti-hyperlipidemic drugs useful in the treatment of hyperlipidemia
in mammals where the symptoms are elevated triglycerides, low HDL
(High density lipoproteins or "good" cholesterol, and elevated
cholesterol. The Fibric Acid amino aicd derivatives are also useful
in reducing LDL (Low density lipoproteins, or "bad"
cholesterol).
[0933] Typical examples of synthesis of L-threonine,
L-hydroxyproline and L-serine esters of Fibric acid derivatives are
shown in the synthetic processes outlined below. These procedures
are applicable to all other compounds of the Fibric acid
derivatives class as well.
Synthesis of Fibric Acid Derivatives Derivatives
[0934] The procedure for the synthesis of the L-serine,
L-threonine, and L-hydroxyproline esters of fenofibric acid is
outlined in Synthetic Sequence section and is exemplary. The
complete procedure and analytical data is given in the Experimental
Section. In general, fenofibric acid (100 g batches) was prepared
from 4-chloro-4'-hydroxybezophenone in accordane with the
procedures in the literature. Fenofibric acid was coupled with the
t-butyl esters of N-Boc protected amino acid (L-serine,
L-threonine, and L-hydroxyproline) using EDC as the coupling agents
and a catalytic amount of DMAP. The protecting groups were removed
at low temperature (5.degree. C., 3-6 days) with a mixture of
hydrochloric acid in acetic acid (1M) with dichloromethane. The
amino acid ester salts of fenofibric acid were purified by
crystallization from ethyl acetate, and dried under high vacuum.
Synthetic Sequence: ##STR132## Synthesis of the L-serine,
L-threonine, and L-hydroxyproline Esters of Fenofibric Acid: a)
Boc-Ser-OtBu, EDC, DMAP, CH.sub.2Cl.sub.2; b) Boc-Thr-OtBu, EDC,
DMAP, CH.sub.2Cl.sub.2; c) Boc-Hyp-OtBu, EDC, DMAP,
CH.sub.2Cl.sub.2; d) HCl, AcOH, CH.sub.2Cl.sub.2. Experimental
Section:
[0935] The synthesis of SPIB00201, SPIB00202 and SPIB00203 was
conducted in one or two batches. Reagents mentioned in the
experimental section were purchased at the highest obtainable
purity from Lancaster, Sigma-Aldrich, Acros, or Bachem, except for
solvents, which were purchased from either Fisher Scientific or
Mallinkrodt. 1) Synthesis of Fenofibric Acid: ##STR133##
[0936] A mixture of 4-chloro-4'-hydroxybezophenone (116 g, 0.500
mole) and sodium hydroxide (120 g, 3.00 mole) in acetone (1 L) was
heated to reflux for 2 hours. The heating was stopped and the
heating source was removed. A mixture of chloroform (179 g, 1.50
mole) in acetone (300 mL) was added drop-wise. The reaction mixture
was stirred overnight without heating. The mixture was heated to
reflux for 8 hours and then allowed to cool to room temperature.
The precipitate was removed by filtration and washed with acetone
(100 mL). The filtrate was concentrated under reduced pressure to
give a brown oil. Water (200 mL) was added to the brown oil and was
acidified (to pH=1) with 1N hydrochloric acid. The precipitate,
which formed was filtered and dried under high vacuum. The
remaining yellow solid (268 g) was recrystallized from toluene in 4
batches (400 mL toluene each). After filtration and drying under
high vacuum, the fenofibric acid (116 g, 73% yield) was obtained as
a light yellow solid.
[0937] .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta.=13.22 (1H, s,
br), 7.72 (4H, d, J=8.4 Hz), 7.61 (2H, d, J=7.8 Hz), 6.93 (2H, d,
J=7.8 Hz), 1.60 (6H, s).
[0938] .sup.13C NMR (75 MHz, DMSO-d.sub.6): .delta.=192.96, 174.18,
159.35, 136.84, 136.12, 131.67, 131.02, 129.12, 128.43, 116.91,
78.87, 25.13.
2) SPIB00201: L-serine-fenofibric Acid Ester
[0939] To a mixture of fenofibric acid (11.6 g, 36.3 mmol),
N-carbobenzyloxy-L-serine t-butyl ester (Boc-Ser-OtBu, 8.62 g, 33.0
mmol), EDC (7.59 g, 39.6 mmol), and DMAP (484 mg, 3.96 mmol) cooled
in an ice-water bath was added anhydrous dichloromethane (150 mL)
dropwise. After the addition was complete, the ice bath was removed
and the reaction mixture was stirred under an argon atmosphere at
room temperature for 20 hours. After 20 hours, the additional
dichloromethane (200 mL) was added, and the solution was washed
with water (2.times.200 mL) and brine (200 mL). After drying over
sodium sulfate and filtration, the solution was concentrated under
reduced pressure. The remaining yellow oil (21.2 g) was purified by
column chromatography on silica gel (400 g, 0.035-0.070 mm, 6 nm
pore diameter), eluting with heptane/ethyl acetate (3:1). After
concentration of the product-containing fractions under reduced
pressure and drying under high vacuum until the weight was
constant, the experiment produced the protected L-serine-fenofibric
acid ester SPIB0020101 (16.2 g, 87% yield) was obtained as a light
yellow oil. ##STR134##
[0940] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.75 (2H, d,
J=9.0 Hz), 7.72 (2H, d, J=9.0 Hz), 7.45 (2H, d, J=8.7 Hz), 6.86
(2H, d, J=8.7 Hz), 5.04 (1H, d, J=6.9 Hz), 4.55-4.42 (3H, m), 1.66
(3H, s), 1.65 (3H, s), 1.43 (9H, s), 1.39 (9H, s).
[0941] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=193.92, 172.99,
168.07, 159.24, 154.87, 138.24, 136.19, 131.94, 131.06, 130.40,
128.41, 117.26, 82.88, 80.13, 79.24, 65.44, 53.44, 28.27, 27.92,
25.70, 25.30.
[0942] To a stirred solution of the protected L-serine-fenofibric
acid ester SPIB0020101 (16.2 g, 28.8 mmol) in anhydrous
dichloromethane (100 mL) cooled to 5.degree. C., under an argon
atmosphere was added a solution of hydrogen chloride in acetic acid
(400 mL, 1M, 400 mmol) drop-wise. The reaction mixture was stirred
for 3 days at 5.degree. C. After three days, the mixture was
concentrated under reduced pressure and dried under high vacuum to
remove acetic acid. To the remaining light yellow oil (24.7 g) was
added ethyl acetate (100 mL). The solution was concentrated and
dried a second time. To the remaining light yellow oil (17.0 g) was
added ethyl acetate (65 mL). The mixture was heated to reflux for 5
minutes and cooled to room temperature. The precipitate was removed
by filtration and dried under high vacuum overnight at room
temperature, then at 43.degree. C. for one hour. The experiment
produced the L-serine-fenofibric acid ester, hydrochloride
SPIB00201 (7.66 g, 60% yield) was obtained as a white solid.
##STR135##
[0943] .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta.=14.12 (1H, s,
br), 8.77 (3H, s, br), 7.72 (4H, m), 7.62 (2H, d, J=8.4 Hz), 6.92
(2H, d, J=9.0 Hz), 4.62 (1H, dd, J=12.0, 4.2 Hz), 4.50 (1H, dd,
J=12.0, 2.4 Hz), 4.41 (1H, m), 1.64 (3H, s), 1.63 (3H, s).
[0944] .sup.13C NMR (75 MHz, DMSO-d.sub.6): .delta.=193.06, 171.70,
168.06, 158.72, 136.93, 136.06, 131.73, 131.09, 129.62, 128.49,
117.64, 79.02, 62.99, 51.11, 25.04, 24.94.
[0945] HPLC analysis:
[0946] 100% purity; r.t.=4.361 min.; 55% TFA (0.1%), 45% ACN; 1
mL/min; 32.3 C, Luna C18, serial # 167917-13; 20 ul inj.,
NB275-49.
[0947] CHN analysis:
[0948] calc.: C, 54.31; H, 4.79; N, 3.17. found: C, 54.37; H, 4.78;
N, 3.12.
[0949] Melting point: 151.degree. C. (dec.)
3) SPIB00202: L-threonine-fenofibric Acid Ester
[0950] To a mixture of fenofibric acid (25.5 g, 79.9 mmol),
N-carbobenzyloxy-L-threonine t-butyl ester (Boc-Thr-OtBu, 20.0 g,
72.6 mmol, prepared by the literature method), EDC (16.7 g, 87.1
mmol), and DMAP (1.06 g, 8.71 mmol) cooled in an ice-water bath was
added anhydrous dichloromethane (200 mL), dropwise. After the
addition was complete, the ice bath was removed and the reaction
mixture was stirred under an argon atmosphere at room temperature
for 20 hours. After 20 hours, additional EDC (1.39 g, 7.26 mmol)
was added, and the reaction mixture was allowed to stir over the
weekend at room temperature under an argon atmosphere. After 4
days, additional dichloromethane (300 mL) was added, and the
solution was washed with water (300 mL) and brine (300 mL). After
drying over sodium sulfate and filtration, the solution was
concentrated under reduced pressure. The remaining yellow oil (53.5
g) was purified by column chromatography on silica gel (500 g,
0.035-0.070 mm, 6 nm pore diameter), eluting with heptane/ethyl
acetate (3:1). After concentration of the product-containing
fractions under reduced pressure and drying under high vacuum until
the weight was constant, the the protected L-threonine-fenofibric
acid ester SPIB0020201 (34.1 g, 82% yield) was obtained as a white
foam. ##STR136##
[0951] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.74 (2H, d,
J=8.4 Hz), 7.72 (2H, d, J=8.4 Hz), 7.45 (2H, d, J=8.4 Hz), 6.87
(2H, d, J=8.4 Hz), 5.47 (1H, m), 4.98 (1H, d, J=9.9 Hz), 4.31 (1H,
d, J=9.9 Hz), 1.65 (3H, s), 1.64 (3H, s), 1.45 (9H, s), 1.42 (9H,
s), 1.22 (3H, d, J=6.3 Hz).
[0952] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=193.94, 172.14,
168.70, 159.26, 155.62, 138.28, 136.18, 131.90, 131.08, 130.37,
128.43, 117.40, 82.70, 80.17, 79.38, 72.02, 57.46, 28.30, 27.99,
26.44, 24.79, 16.90.
[0953] To a stirred solution of the protected
L-threonine-fenofibric acid ester SPIB0020201 (34.1 g, 59.2 mmol)
in anhydrous dichloromethane (100 mL) cooled to 5.degree. C., under
an argon atmosphere was added a solution of hydrogen chloride in
acetic acid (600 mL, 1M, 600 mmol) drop-wise. The reaction mixture
was kept for 6 days at 5.degree. C. The mixture was concentrated
under reduced pressure and dried under high vacuum to remove acetic
acid. To the remaining white solid (45.8 g) was added ethyl acetate
(500 mL). The mixture was heated to reflux for 10 minutes and
cooled to room temperature. The precipitate was removed by
filtration and dried under high vacuum overnight at room
temperature, yielding the L-threonine-fenofibric acid ester,
hydrochloride SPIB00202 (26.3 g, 97% yield) as a white solid.
##STR137##
[0954] .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta.=14.10 (1H, s,
br), 8.84 (3H, s, br), 7.73 (4H, m), 7.63 (2H, d, J=8.1 Hz), 6.89
(2H, d, J=8.7 Hz), 5.44 (1H, m), 4.31 (1H, s), 1.64 (3H, s), 1.62
(3H, s), 1.38 (3H, d, J=6.3 Hz).
[0955] .sup.13C NMR (75 MHz, DMSO-d.sub.6): .delta.=193.04, 171.00,
168.13, 158.76, 136.90, 136.08, 131.70, 131.06, 129.49, 128.48,
117.41, 78.99, 69.40, 55.21, 25.59, 24.22, 16.06.
[0956] HPLC analysis:
[0957] 98.59% purity; r.t.=4.687 min.; 55% TFA (0.1%), 45% ACN; 1
mL/min; 32.3 C, Luna C18, serial # 167917-13; 20 ul inj., NB275-49,
DAD1 B, Sig=210.4, Ref=550,100.
[0958] CHN analysis:
[0959] calc.: C, 55.27; H, 5.08; N, 3.07. found: C, 54.98; H, 5.13;
N, 3.03.
[0960] Melting point: 160.5.degree. C. (dec.)
4) SPIB00203: L-hydroxyproline-fenofibric Acid Ester
[0961] To a mixture of fenofibric acid (24.9 g, 78.1 mmol),
N-carbobenzyloxy-L-hydroxyproline t-butyl ester (Boc-Hyp-OtBu, 20.4
g, 71.0 mmole, prepared in accordance with the procedure in the
literature), EDC (16.3 g, 85.2 mmol), and DMAP (1.04 g, 8.52 mmol)
cooled in an ice-water bath was added anhydrous dichloromethane
(200 mL) dropwise. After the addition was complete, the ice bath
was removed and the reaction mixture was stirred under an argon
atmosphere at room temperature for 20 hours. After 20 hours,
additional EDC (1.63 g, 8.52 mmol) was added and the experiment was
allowed to stir over the weekend at room temperature under an argon
atmosphere. After 4 days, the resulting solution was washed with
water (200 mL) and brine (200 mL). After drying over sodium sulfate
followed by filtration, the solution was concentrated under reduced
pressure. The remaining yellow oil (49.4 g) was purified by column
chromatography on silica gel (500 g, 0.035-0.070 mm, 6 nm pore
diameter), eluting with heptane/ethyl acetate (2:1). After
concentration of the product containing fractions under reduced
pressure and drying under high vacuum until the weight was
constant, the protected L-hydroxyproline-fenofibric acid ester
SPIB0020301 (26.4 g, 63% yield) was obtained as a colorless oil.
##STR138##
[0962] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=7.76 (2H, d,
J=8.1 Hz), 7.73 (2H, d, J=8.1 Hz), 7.46 (2H, d, J=8.1 Hz), 6.84
(2H, d, J=8.1 Hz), 5.32 (1H, m), 4.13 (0.38H, t, J=7.8 Hz), 4.00
(0.62H, t, J=7.8 Hz), 3.67 (1.62H, m), 3.46 (0.38H, d, J=12.6 Hz),
2.29 (1H, m), 2.15 (1H, m), 1.68 (3H, s), 1.66 (3H, s), 1.44-1.38
(18H, m).
[0963] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.=193.88, 172.98,
171.14, 159.25, 153.48, 138.23, 136.16, 131.99, 131.08, 130.36,
128.44, 117.03, 116.91, 81.48, 80.32, 80.20, 79.19, 74.03, 73.26,
58.23, 51.88, 51.58, 36.33, 35.31, 31.92, 28.29, 28.00, 25.89,
24.95.
[0964] To a stirred solution of the protected
L-hydroxyproline-fenofibric acid ester SPIB0020301 (26.0 g, 44.2
mmol) in anhydrous dichloromethane (100 mL) cooled to 5.degree. C.,
under an argon atmosphere was added a solution of hydrogen chloride
in acetic acid (450 mL, 1M, 450 mmol) drop-wise. The reaction
mixture stirred for 4 days at 5.degree. C. After four days the
mixture was concentrated under reduced pressure and dried under
high vacuum to remove acetic acid. To the remaining yellow oil
(31.5 g) was added ethyl acetate (200 mL). The mixture was
sonicated and then concentrated under reduced pressure and dried
under high vacuum. To the remaining white solid (23.2 g) was added
ethyl acetate (300 mL). The ethyl acetate mixture was heated to
reflux for 10 minutes, and cooled to room temperature. The
precipitate was removed by filtration and dried under high vacuum
overnight at room temperature. L-hydroxyproline-fenofibric acid
ester, hydrochloride SPIB00203 (15.8 g, 76% yield) was obtained as
a white solid. ##STR139##
[0965] .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta.=14.07 (1H, s,
br), 10.75 (1H, s, br), 9.40 (1H, s, br), 7.71 (4H, d, J=8.1 Hz),
7.60 (2H, d, J=8.1 Hz), 6.96 (2H, d, J=8.1 Hz), 5.42 (1H, m), 4.24
(1H, t, J=9.0 Hz), 3.61 (1H, dd, J=13.2, 4.2 Hz), 3.28 (1H, d,
J=13.2 Hz), 2.35 (2H, m), 1.66 (3H, s), 1.64 (3H, s).
[0966] .sup.13C NMR (75 MHz, DMSO-d.sub.6): .delta.=193.00, 171.52,
169.14, 158.81, 136.87, 136.09, 131.81, 131.05, 129.48, 128.46,
117.28, 78.99, 73.79, 57.54, 50.23, 34.13, 25.69, 24.49.
[0967] HPLC analysis:
[0968] 100% purity; r.t.=8.369 min.; 60% DIUF water (0.1% TFA)/40%
acetonitrile; 1 mL/min; 36.4 C; Luna C18, 5 u column (serial #
191070-3), 4.6.times.250 mm; 20 ul injection; DAD1 A, Sig=210.4,
Ref=550,100.
[0969] HPLC-MS (ESI): calculated: M.sup.+=431; found M+H=432.3
[0970] Melting point: 187.5.degree. C. (dec.)
[0971] Solubility of the above esters were determined in water at
room temperature by dissolving an excess of each of the drug and
permitting each to settle for a few hours. The resulting solutions
were centrifuged at 1500 rpm for 3 min and the supernatant liquid
was analyzed. By providing the memory of Sikihn, Esq. esters posses
solubility in water in excess of 50 mg/mL.
EXPERIMENTAL
[0972] Rats were checked for time zero triglyceride level in blood.
Then the rats were set on high sugar diet, such as 30% surcorse in
water for 1 week. Then at the end of 1 week, rats were tested for
triglycerides, and were put on normal diet. From day 7-14 the rats
were administered either test or control drug. Triglycerides were
again tested on the 14.sup.th day in rat blood.
[0973] In the Fenofibrate (control) vs L-Serine Ester of Fenofibric
acid (test drug), 3 rats each for each of the drug and control at
equivalent doses of 50, 100 and 200 mg/kg were tested.
[0974] The results are shown below in Tables 35 and 36.
TABLE-US-00038 TABLE 35 SUMMARY - DOSE RANGE FINDING STUDY -
HYPOLIPIDEMIC PROPERTY - FENOFIBRATE AND ITS FORMULATION Test
Substance: L-Serine Ester of Fenofibric Acid Vehicle: 1% Tween 80
in milli Q - water Dose. Triglycerides (mg/dl) Test Item Mg/kg)
Animal No. Day zero Day 7 Day 14 Vehicle 0 1 81 168 121 2 88 171
222 3 114 133 162 Reference control 50 4 95 157 101 Fenofibrate 5
92 228 76 6 80 150 73 100 7 110 204 62 8 115 195 69 9 96 167 93 200
10 144 90 48 11 56 106 51 12 58 125 38 L-Serine Ester of 50 13 88
148 86 Fenofibric Acid 14 94 145 86 15 100 127 73 100 16 109 -- 46
17 129 100 69 18 71 183 47 200 19 74 240 83 20 81 158 61 21 42 77
46
[0975] TABLE-US-00039 TABLE 36 Anti-lipidemic Effects of Fenofibric
Acid and its Derivatives Absolute % % Change Absolute Change Change
from Change From from Dose day 0 from day 7 day 0 day 7 Vehicle
mg/kg 50.4 -5.6 83.17% -4.80% Fen-S 25 -54 -90 -61.64% -72.82%
Ester 50 -31.4 -84.2 -44.10% -67.90% 100 -20.8 -72 -33.23% -63.27%
Fen-T 25 -18.2 -36.4 -23.21% -37.68% Ester 50 -23.8 -77.6 -35.00%
-63.71% 100 -63.8 -88.4 -68.45% -75.04% Fen-HP 25 -16 -47.8 -32.92%
-59.45% Ester 50 -35.8 -70.8 -49.31% -65.80% 100 -3.4 -112 -7.52%
-72.82% Fenofibrate 25 -10.8 -51 -15.21% -45.86% 50 -13.4 -87.6
-22.95% -66.06% 100 -40.8 -71.6 -61.26% -73.51%
[0976] While all of the esters were active and showed efficacy,
there were important distinguishing factors between various amino
acid derivatives and Fenofibrate. For example, dose dependent
decrease in triglycerides were noted with the L-Threonine ester,
and also maximum decrease from baseline level and treatment level
were also noted for this compound. Thus Fenofibric Acid-L-Threonine
ester had overall superior anti-hyperlipidemic properties.
[0977] From the above results, it can be concluded that both the
highly water soluble serine ester, L-threonine ester and
hydroxyproline ester effectively performed compared to
fenofibrate.
[0978] There are a number of screening tests to determine the
utility of the amino acid derivatives created according tio the
disclosed methods. These include both in vitro and in vivo
screening methods.
[0979] The in vitro methods include acid/base hydrolysis of the
amino acid derivatives, e.g., hydrolysis in pig pancreas hydrolysis
in rat intestinal fluid, hydrolysis in human gastric fluid,
hydrolysis in human intestinal fluid, and hydrolysis in human blood
plasma. These assays are dscribed in Simmons, D M, Chandran, V R
and Portmann, G A, Danazol Amino Acid Derivatives: In Vitro and In
Situ Biopharmaaceutical Evaluation, Drug Development and Industrial
Pharmacy, Vol 21, Issue 6, Page 687, 1995, the contents of all of
which are incorporated by reference.
[0980] The amino acid derivatives of Fibric Acid of the present
invention are effective in treating diseases or conditions in which
Fibric acid derivatives normally are used. The amino acid
derivatives disclosed herein are transformed within the body to
potentially release the active compound, although the active agent
in vivo may be the intact amino acid derivative of fibric acid. In
addition, the amino acid derivatives of fibric acid also enhance
the therapeutic benefits of the Fibric acid compounds by reducing
or eliminating biopharmaceutical and pharmacokinetic barriers
associated with each of them. However it should be noted that these
amino acid derivatives themselves will have sufficient activity
without releasing any active drug in the mammals.
[0981] Thus, the amino acid derivatives of the present invention
enhance the therapeutic benefits by removing biopharmaceutical and
pharmacokenetic barriers of existing drugs.
[0982] Furthermore, the amino acid derivatives are easily
synthesized in high yields using reagents which are readily and
commercially available.
[0983] In the formula hereinabove, it is to be understood that the
AA has the following definition in the following context unless
indicated to the contrary ##STR140## AA in this definition refers
to the amino acid residue without an amino group either on the main
chain or the side chain. ##STR141## AA in this definition is an
amino acid residue less the hydroxy group on the side chain.
##STR142## AA refers to an amino acid group without the carboxy
group, either on the main chain or side group. 4) OAA--This is a
ester bond between the hydroxy group of the drug and the carboxy
group of the amino acid either on the main chain or side chain.
Thus, as written OAA is ##STR143## wherein R.sub.O is the side
chain amino acid as defined hereinabove.
[0984] Alternatively, it may refer to an ester bond between the
carboxy group of the drug and the hydroxy group on the side chain
of those amino acids which have a hydroxy group thereon such as
threonine, serine, hydroxyproline, tyrosine and the like. The
hydroxy group forms part of the ester linkage which is depicted
hereinabove with O. Thus, as written, the AA refers to an amino
acid with a hydroxy group on the side chain, but as depicted as
OAA, the AA is without the hydroxy group since the oxygen atom is
depicted in the formula.
[0985] As used herein, the term "amino acid derivative" or
equivalent term thereto refers to amino acid moiety covalently
bonded to the drug having a functional group thereon consisting of
hydroxy, amino, carboxy and acylating derivatives of carboxy.
[0986] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *